pLKO5.sgRNA.EFS.tRFP

LOCUS       pLKO5.sgRNA.EFS.tRFP                7408 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  3rd generation Lentiviral Vector for Sp sgRNA delivery without 
            SpCas9, tagRFP, EFS Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO5.sgRNA.EFS.tRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 7408)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7408
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        268..298
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /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)."
     primer_bind     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     365..385
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        367..385
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        413..639
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             640..820
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    867..992
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1485..1718
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1903..1947
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2125..2365
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2125..2145
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2296..2315
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2392..2467
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2515..2632
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2701..2912
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2932..2941
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2938..3651
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    3673..4261
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3726..3746)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4144..4155)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4333..4566
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4644..4765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4681..4700)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4735..4754
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4805..4940
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4867..4886
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4960..4979)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4961..4979)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4989..5006)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4989..5005)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4998..5020)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5147..5602
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5234..5253)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5444..5465
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5628..5732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5733..6593
                     /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     complement(5951..5970)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6764..7352
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7253..7272
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      361 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
      421 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
      481 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
      541 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
      601 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
      661 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      721 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      781 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      841 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
      901 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
      961 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1021 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1081 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1141 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1201 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1261 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1321 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1381 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1441 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1501 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1561 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1621 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1681 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1741 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1801 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1861 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1921 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1981 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     2041 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     2101 ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat
     2161 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2221 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2281 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2341 gctttatata tcttgtggaa aggacgaaac accggagacg tgaccgtctc tgttttagag
     2401 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     2461 tcggtgcttt tttgaattct agatcttgag acaaatggca gtattcatcc acaattttaa
     2521 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2581 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2641 ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt gcccgtcagt
     2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2881 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc tgccaccatg
     2941 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
     3001 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     3061 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     3121 ctggctacca gcttcatgta cggcagcaga accttcatca accacaccca gggcatcccc
     3181 gacttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     3241 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     3301 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca
     3361 ctcggctggg aggccaacac cgagatgctg taccccgctg acggcggcct ggaaggcaga
     3421 agcgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca
     3481 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggaccac
     3541 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct
     3601 gtggccagat actgcgacct ccctagcaaa ctggggcaca aacttaattg aggtaccacg
     3661 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3721 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3781 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3841 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3901 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3961 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4021 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     4081 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     4141 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4201 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4261 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4321 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     4381 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4441 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4501 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4561 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4621 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4741 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4801 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4861 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4921 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4981 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5041 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5101 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     5161 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5221 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5281 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5341 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5401 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5461 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5521 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5581 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5641 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5701 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5761 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5821 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5881 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5941 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6001 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6061 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6121 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6181 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6241 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6301 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6361 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6421 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6481 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6541 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6601 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6661 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6721 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6781 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6841 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6901 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6961 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7021 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7081 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7141 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7201 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7261 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7321 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7381 tcctggcctt ttgctggcct tttgctca
//