lentiSAMv2

LOCUS       lentiSAMv2               13500 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  lenti sgRNA cloning backbone with MS2 loops at tetraloop and 
            stemloop 2, dCas9-VP64, and blast resistance marker. Contains BsmBI 
            sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    lentiSAMv2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13500)
  AUTHORS   Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham
            MD, Sanjana NE, Zhang F
  TITLE     Genome-scale CRISPR-Cas9 knockout and transcriptional activation 
            screening.
  JOURNAL   Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. 
            Epub 2017 Mar 23.
  PUBMED    28333914
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|biofeng.com|
COMMENT     VNTNAME|lentiSAMv2|
FEATURES             Location/Qualifiers
     source          1..13500
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(113..133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        118..447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      298..433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     360..379
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        495..542
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             561..935
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1065..1186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1102..1121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1156..1175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1235..1251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1235..1251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1248..1270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1259..1275
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(1283..1313)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1328..1349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1466..1483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1637..2225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1717..1736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2396..3256)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3019..3038
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3257..3361)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3436..3455)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3627..4006
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4008..4206
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4161..4181
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4224..4404
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4451..4576
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5069..5302
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5829..5946
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5996..6236
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     5996..6016
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6167..6186
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6282..6300
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        6352..6370
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        6457..6668
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             6685..6732
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6733..10833
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             10888..10908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10933..11082
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             11104..11157
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             11158..11556
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    11581..12169
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11634..11654)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12052..12063)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12172..12188)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12173..12189)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12694..12874
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12900..12917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12906..13130
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      13176..104
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13263..13282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13473..13494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
       61 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
      121 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
      181 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
      241 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
      301 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
      361 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
      421 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      481 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
      541 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
      601 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
      661 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
      721 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
      781 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
      841 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
      901 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
      961 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     1021 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     1081 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1141 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     1201 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1261 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1321 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1441 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1501 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1561 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1621 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1681 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1741 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1801 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1861 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1921 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1981 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2041 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2101 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2161 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2221 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2281 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2341 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2401 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2461 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2521 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2581 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2641 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2701 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2761 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2821 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2881 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2941 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3001 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3061 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3121 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3181 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3241 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3301 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3361 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     3421 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     3481 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     3541 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     3601 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     3661 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3721 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3781 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3841 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3901 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3961 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     4021 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     4081 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     4141 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     4201 aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4261 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     4321 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4381 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     4441 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     4501 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     4561 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     4621 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     4681 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     4741 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     4801 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     4861 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     4921 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     4981 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     5041 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     5101 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     5161 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     5221 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     5281 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     5341 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     5401 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     5461 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     5521 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     5581 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     5641 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     5701 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     5761 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     5821 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     5881 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     5941 aattttcggg tttattacag ggacagcaga gatccagttt ggttaattag ctagcgaggg
     6001 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     6061 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     6121 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     6181 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     6241 caccggagac gggataccgt ctctgtttta gagctaggcc aacatgagga tcacccatgt
     6301 ctgcagggcc tagcaagtta aaataaggct agtccgttat caacttggcc aacatgagga
     6361 tcacccatgt ctgcagggcc aagtggcacc gagtcggtgc tttttttgga tcctaggtct
     6421 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     6481 tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg
     6541 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     6601 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     6661 gaacacaggc gtacggccac catgaaaagg ccggcggcca cgaaaaaggc cggccaggca
     6721 aaaaagaaaa aggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc
     6781 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     6841 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     6901 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     6961 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     7021 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     7081 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     7141 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     7201 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     7261 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     7321 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     7381 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     7441 ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     7501 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     7561 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     7621 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     7681 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     7741 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     7801 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     7861 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     7921 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     7981 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     8041 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     8101 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     8161 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     8221 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     8281 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     8341 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     8401 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     8461 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     8521 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     8581 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     8641 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     8701 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     8761 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     8821 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     8881 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     8941 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     9001 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     9061 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     9121 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     9181 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     9241 gtggaccaca tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     9301 accagaagcg acaaggcccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     9361 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     9421 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     9481 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     9541 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     9601 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     9661 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     9721 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     9781 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     9841 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     9901 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     9961 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
    10021 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
    10081 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
    10141 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
    10201 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
    10261 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
    10321 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
    10381 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
    10441 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
    10501 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
    10561 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
    10621 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
    10681 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
    10741 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
    10801 acacggatcg acctgtctca gctgggaggc gacagcgctg gaggaggtgg aagcggagga
    10861 ggaggaagcg gaggaggagg tagcggacct aagaaaaaga ggaaggtggc ggccgctgga
    10921 tccggacggg ctgacgcatt ggacgatttt gatctggata tgctgggaag tgacgccctc
    10981 gatgattttg accttgacat gcttggttcg gatgcccttg atgactttga cctcgacatg
    11041 ctcggcagtg acgcccttga tgatttcgac ctggacatgc tgattaactg tacaggcagt
    11101 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
    11161 gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac
    11221 agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc
    11281 atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg
    11341 ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat
    11401 gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat
    11461 cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt
    11521 gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt
    11581 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
    11641 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
    11701 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
    11761 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
    11821 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
    11881 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
    11941 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
    12001 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
    12061 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
    12121 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
    12181 cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat
    12241 gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct
    12301 caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
    12361 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg
    12421 tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggg
    12481 atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag
    12541 gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg
    12601 atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat
    12661 cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga
    12721 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
    12781 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
    12841 agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc
    12901 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
    12961 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
    13021 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
    13081 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
    13141 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
    13201 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
    13261 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
    13321 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
    13381 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
    13441 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
//