LOCUS pT3TS-nCas9n 7332 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pT3TS-nCas9n
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7332)
AUTHORS Jao LE, Wente SR, Chen W
TITLE Efficient multiplex biallelic zebrafish genome editing using a
CRISPR nuclease system.
JOURNAL Proc Natl Acad Sci U S A. 2013 Aug 5.
PUBMED 23918387
REFERENCE 2 (bases 1 to 7332)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7332
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..19
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
5'UTR 44..86
/label=Xenopus globin 5'-UTR
/note="translational enhancer from the 5'-UTR of the major
beta-globin gene of Xenopus laevis"
regulatory 102..111
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 120..140
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 147..4247
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes MGAS15252 Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
RBS 4160..4168
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4263..4283
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
3'UTR 4308..4433
/label=Xenopus globin 3'-UTR
/note="3'-UTR of the major beta-globin gene of Xenopus
laevis"
primer_bind complement(4520..4539)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(4521..4539)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4546..4563)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4546..4562)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4555..4577)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin complement(4704..5159)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4841..4862)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5053..5072
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 5186..5290
/gene="bla"
/label=AmpR promoter
CDS 5291..6151
/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(5509..5528)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6322..6910
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6811..6830
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7064..7081
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 7198..7219
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7234..7264
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7272..7288
/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 7277..7299
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 7296..7312
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 7296..7312
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg gcttctccac
121 ctaagaagaa gagaaaggtg ggaagcatgg ataagaagta tagcatcggc ctggatattg
181 gaactaactc cgtgggttgg gcagtgatta cagacgacta caaggtccct agcaagaaat
241 ttaaggtgct gggtaacacc gacaggcaca gcatcaagaa aaatctgatt ggagccctgc
301 tgttcggttc tggagagact gccgaagcaa cacgcctgaa aagaacagca agaaggcgct
361 ataccagaag gaagaataga atctgttacc tgcaggagat tttctctaac gaaatggcta
421 aggtggacga ttcattcttt cataggctgg aggaaagttt cctggtcgag gaagataaga
481 aacacgagcg ccatcctatc tttggaaaca ttgtggacga ggtcgcctat cacgaaaaat
541 acccaaccat ctatcatctg cgcaagaaac tggctgactc tactgataaa gccgacctga
601 gactgatcta tctggctctg gcccacatga ttaagttcag gggtcatttt ctgatcgagg
661 gcgatctgaa ccccgacaat tccgatgtgg acaagctgtt catccagctg gtccagattt
721 acaatcagct gtttgaggaa aaccctatta atgcttccag agtggacgca aaagctatcc
781 tgtcagccag gctgtccaag tcacgcagac tggagaacct gattgcacag ctgcccggag
841 aaaagaggaa cggtctgttt ggaaatctga tcgctctgag tctgggcctg actcctaact
901 tcaaaagcaa ttttgatctg gctgaggacg ccaaactgca gctgtcaaag gacacatatg
961 acgatgacct ggataacctg ctggcacaga tcggagatca gtacgctgac ctgttcctgg
1021 ctgccaaaaa tctgtccgac gcaatcctgc tgtcagatat tctgagagtg aacagcgaga
1081 ttacaaaagc tcctctgagt gccagcatga tcaagagata tgacgagcac catcaggatc
1141 tgaccctgct gaaggctctg gtcaggcagc agctgccaga gaagtacaag gaaattttct
1201 ttgatcagtc caagaacggc tacgccggtt atatcgacgg aggcgcatca caggaggaat
1261 tctacaagtt tatcaaacct attctggaga agatggacgg aactgaggaa ctgctggtga
1321 aactgaatag agaggacctg ctgaggaagc agcgcacatt tgataacggt tccatcccac
1381 accagattca tctgggagag ctgcacgcta tcctgaggcg ccaggaagac ttctacccct
1441 ttctgaaaga taaccgcgag aagatcgaaa aaattctgac cttcagaatc ccttactatg
1501 tgggtccact ggctcgcgga aacagcagat ttgcctggat gactcgcaaa tccgaggaaa
1561 ccattactcc ttggaacttc gaggaagtgg tcgataaggg cgcctctgca cagtccttca
1621 tcgagagaat gactaatttt gacaaaaacc tgcccaatga gaaagtgctg cctaagcact
1681 ccctgctgta cgagtatttc actgtctata acgaactgac aaaggtgaaa tacgtcaccg
1741 agggcatgag aaagccagcc ttcctgtcag gagagcagaa gaaagcaatc gtggatctgc
1801 tgtttaaaac caacaggaaa gtgactgtca agcagctgaa ggaggactac ttcaagaaaa
1861 ttgaatgctt cgattccgtg gagatcagcg gagtcgaaga cagatttaac gcaagcctgg
1921 gcgcttacca cgatctgctg aagatcatta aggataaaga cttcctggac aacgaggaaa
1981 atgaggatat cctggaagac attgtgctga cactgaccct gtttgaggac agaggaatga
2041 tcgaggaaag actgaaaacc tatgctcatc tgttcgatga caaggtgatg aaacagctga
2101 agagaaggcg ctacactggc tggggtagac tgagcaggaa gctgatcaac ggcattaggg
2161 ataaacagtc aggaaagaca atcctggact ttctgaaaag tgatggcttc gccaaccgca
2221 attttatgca gctgattcac gatgacagtc tgaccttcaa agaggacatc cagaaggctc
2281 aggtgtctgg acagggccac tccctgcatg agcagattgc aaacctggct ggaagcccag
2341 ccatcaagaa aggcattctg cagacagtga aaatcgtcga tgagctggtg aaagtcatgg
2401 gccataagcc cgaaaacatc gtgattgaga tggctcgcga aaatcagaca acccagaagg
2461 gtcagaagaa cagtagagag aggatgaaaa gaatcgagga aggcattaag gagctgggta
2521 gccagatcct gaaagagcac ccagtggaaa acacacagct gcagaatgag aagctgtatc
2581 tgtactatct gcagaatgga agagatatgt acgtggacca ggagctggat attaacaggc
2641 tgtctgatta cgacgtggat catatcgtcc cccagagttt catcaaagat gacagcattg
2701 acaacaaggt gctgaccagg tccgacaaaa acagaggaaa atcagataat gtccctagtg
2761 aggaagtggt caagaaaatg aagaactact ggagacagct gctgaatgcc aaactgatca
2821 ctcagaggaa gtttgataac ctgacaaaag cagagcgcgg tggactgtca gaactggaca
2881 aagctggatt catcaagagg cagctggtgg aaacacgcca gatcactaaa cacgtcgcac
2941 agattctgga tagtcgcatg aacacaaagt acgatgagaa tgacaaactg atcagagaag
3001 tgaaggtcat taccctgaag agtaaactgg tcagcgactt taggaaagat ttccagtttt
3061 ataaggtccg cgagattaac aattatcacc atgcccatga cgcatacctg aacgccgtgg
3121 tcggtaccgc actgatcaag aaatacccaa aactggagag cgaattcgtg tacggagact
3181 ataaggtgta cgatgtcaga aaaatgatcg ccaagtccga gcaggaaatt ggaaaagcta
3241 ctgccaagta tttcttttac tcaaacatca tgaatttctt taagacagag atcaccctgg
3301 ccaatggaga aatccgcaaa aggcccctga ttgagacaaa cggagagaca ggcgaaatcg
3361 tgtgggacaa aggcagagat tttgcaaccg tgaggaaggt cctgagcatg cctcaagtga
3421 atatcgtcaa gaaaactgag gtgcagacag gcggtttctc aaaagaaagt attctgccaa
3481 aacgcaactc tgataagctg atcgctagaa agaaagactg ggaccctaag aagtatggag
3541 gctttgactc tcccactgtg gcatactccg tcctggtggt cgctaaggtg gagaagggca
3601 aaagcaagaa actgaaatct gtcaaggagc tgctgggtat cacaattatg gagagaagct
3661 ctttcgagaa gaacccaatc gattttctgg aggccaaagg ttataaggaa gtgaagaaag
3721 acctgatcat taaactgccc aagtacagtc tgtttgagct ggaaaacggc aggaaacgca
3781 tgctggcaag cgctggagag ctgcagaaag gcaatgaact ggccctgcct tctaagtacg
3841 tgaacttcct gtatctggca agccactacg agaagctgaa aggatctcca gaggataacg
3901 aacagaaaca gctgtttgtg gagcagcaca agcattatct ggacgagatc attgaacaga
3961 ttagcgagtt ctctaaaaga gtgatcctgg ccgacgcaaa tctggataag gtcctgtctg
4021 cttacaacaa acacagagat aagcccatca gggagcaggc cgaaaatatc attcatctgt
4081 tcactctgac aaacctgggc gcacctgcag ctttcaagta cttcgacact acaatcgata
4141 gaaagaggta cacctccact aaggaggtgc tggacgctac actgatccat cagagtatta
4201 ccggcctgta cgaaacaagg attgacctgt ctcagctggg tggcgactca ccggtgagat
4261 ctcctaagaa gaagagaaag gtgtgaccgc ggatctggtt accactaaac cagcctcaag
4321 aacacccgaa tggagtctct aagctacata ataccaactt acactttaca aaatgttgtc
4381 ccccaaaatg tagccattcg tatctgctcc taataaaaag aaagtttctt cacattctaa
4441 aaaaaaaaaa aaaaaaaaaa aaaaaaancc cccccccccc cctgcagggc caagtcggcc
4501 tctagaggta cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
4561 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
4621 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
4681 gcgcagcctg aatggcgaat ggcaaattgt aagcgttaat attttgttaa aattcgcgtt
4741 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
4801 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
4861 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
4921 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
4981 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
5041 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
5101 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
5161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
5221 ttcaaatatg tatctgctca tgagacaata accctgataa atgcttcaat aatattgaaa
5281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
5341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
5401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
5461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
5521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
5581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
5641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
5701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
5761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
5821 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
5881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
5941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
6001 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
6061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
6121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
6181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
6241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
6301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
6361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
6421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
6481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
6541 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
6601 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
6661 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
6721 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
6781 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
6841 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
6901 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
6961 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
7021 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
7081 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
7141 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
7201 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
7261 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
7321 cgccaagctc ga
//
