LOCUS       Exported                7487 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector for encoding a human codon-optimized SpCas9 driven by EFs
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    AAV_Efs_hSpCas9_NLS_FLAG-SV40
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7487)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 7487)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7487
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      106..561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(193..212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     403..424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(578..597)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     697..719
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(757..775)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        843..947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             948..1808
                     /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(1166..1185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1979..2567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2468..2487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2629..2758
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2814..3025
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3044..3053
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3050..7153
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7154..7201
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7202..7225
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    7235..7369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7272..7291)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7326..7345
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7378..31
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7378..20
                     /label=AAV2 ITR
ORIGIN
        1 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
       61 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
      121 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      181 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      241 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      301 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
      361 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
      421 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
      481 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
      541 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
      601 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
      661 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
      721 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
      781 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
      841 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
      901 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
      961 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1021 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1081 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1141 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1201 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1261 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1321 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1381 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1441 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     1501 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1561 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     1621 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     1681 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1741 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1801 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     1861 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1921 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1981 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2041 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2101 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2161 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2221 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2281 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2341 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2401 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2461 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2521 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2581 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc
     2641 tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc
     2701 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc
     2761 ggcctctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
     2821 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
     2881 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     2941 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     3001 caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa
     3061 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     3121 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     3181 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     3241 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     3301 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     3361 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     3421 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     3481 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     3541 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     3601 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     3661 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     3721 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     3781 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     3841 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     3901 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     3961 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     4021 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     4081 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     4141 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     4201 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     4261 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     4321 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     4381 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     4441 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     4501 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     4561 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     4621 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     4681 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     4741 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     4801 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     4861 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     4921 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     4981 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     5041 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     5101 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     5161 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     5221 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     5281 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     5341 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     5401 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     5461 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     5521 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     5581 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     5641 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     5701 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     5761 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     5821 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     5881 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     5941 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     6001 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     6061 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     6121 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     6181 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     6241 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     6301 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     6361 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     6421 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     6481 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     6541 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     6601 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     6661 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     6721 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     6781 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     6841 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     6901 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     6961 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     7021 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     7081 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     7141 gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa
     7201 agattacaaa gacgatgacg ataagggatc ctaaaacttg tttattgcag cttataatgg
     7261 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     7321 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg cggccgcagg
     7381 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     7441 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctca
//
