LOCUS       sequence-183605-        7881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1-targeting expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7881)
  AUTHORS   Chen X, Janssen JM, Liu J, Maggio I, 't Jong AEJ, Mikkers HMM, 
            Goncalves MAFV
  TITLE     In trans paired nicking triggers seamless genome editing without 
            double-stranded DNA cutting.
  JOURNAL   Nat Commun. 2017 Sep 22;8(1):657. doi: 10.1038/s41467-017-00687-1.
  PUBMED    28939824
REFERENCE   2  (bases 1 to 7881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-09-22"; volume: "8(1):657. doi"; pages: " 
            10.1038/s41467-017-00687-1"
COMMENT     Sequence Label: AV11_pDonor.EP.S1
FEATURES             Location/Qualifiers
     source          1..7881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    235..256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(271..276,277..294,295..301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    309..325
                     /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     314..336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     333..349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        370..388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    1160..1963
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        1974..2483
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2403..2421
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2506..3102
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2506..2525)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3002..3022
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3112..3165
                     /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"
     regulatory      3172..3181
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3178..3180,3181..3183,3184..3897)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3178..3180,3181..3183,3184..3894)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3223..3244)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3484..3503)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3831..3852
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3924..3941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3930..4154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4162..4998
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5791..5810)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5792..5810)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5817..5833)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5900..5917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6071..6659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6151..6170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6830..7621,7622..7690))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7453..7472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7691..7795)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7863..7881
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctttcgtct tcaagaattg gaaacagcta tgaccatgat tacgccaagc tcgaaattac
       61 ccctcactaa agggaacaaa gctggtacga ggacaggctg gagccatggg catggctact
      121 caagctgatt tgatggagtt ggacatggcc atggctggtg accacgtcgt ggaatgcctt
      181 cgaattcagc acctgcacat gggacgtcga cctgaggtaa ttagtaccgc ggcctaatgt
      241 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      301 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      361 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctccacc gcggtggcgg
      421 cccgccgagc tcgaccccgc cgcgccgccg cccgcccgcg ccgtgctgga ctccaccaac
      481 gccgacggta tcagcgccct gcaccaggtc agcgcccccc gcccggcgtc tcccggggcc
      541 aggtccaccc tctgctgcgc cacctggggc atcctccttc cccgttgcca gtctcgatcc
      601 gccccgtcgt tcctggccct gggctttgcc accctatgct gacaccccgt cccagtcccc
      661 cttaccattc cccttcgacc accccacttc cgaattggag ccgcttcaac tggccctggg
      721 cttagccact ctgtgctgac cactctgccc caggcctcct taccattccc cttcgaccta
      781 ctctcttccg cattggagtc gctttaactg gccctggctt tggcagcctg tgctgaccca
      841 tgcagtcctc cttaccatcc ctccctcgac ttcccctctt ccgatgttga gcccctccag
      901 ccggtcctgg actttgtctc cttccctgcc ctgccctctc ctgaacctga gccagctccc
      961 atagctcagt ctggtctatc tgcctggccc tggccattgt cactttgcgc tgccctcctc
     1021 tcgcccccga gtgcccttgc tgtgccgccg gaactctgcc ctctaacgct gccgtctctc
     1081 tcctgagtcc ggaccacttt gagctctact ggcttctgcg ccgcctctgg cccactgttt
     1141 ccccttccca ggcaggtcct gctttctctg acctgcattc tctcccctgg gcctgtgccg
     1201 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc
     1261 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc
     1321 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc
     1381 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc
     1441 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc
     1501 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt
     1561 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt
     1621 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg
     1681 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg
     1741 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc
     1801 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg
     1861 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg
     1921 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagcttc cacggggttg
     1981 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     2041 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc
     2101 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     2161 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     2221 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     2281 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     2341 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     2401 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     2461 cgaatcaccg acctctctcc ccaccgggta cgtcgctagc ccaccatgac cgagtacaag
     2521 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc
     2581 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg
     2641 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     2701 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     2761 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     2821 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     2881 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     2941 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     3001 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     3061 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg agagggcaga
     3121 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt cgccaccatg
     3181 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3241 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3301 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3361 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3421 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3481 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3541 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3601 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3661 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3721 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3781 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3841 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc
     3901 ggccgcgtcg agtctaggat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3961 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4021 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4081 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4141 ggtgggctct atggaagctt tactagggac aggattggtg acagaaaagc cccatcctta
     4201 ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat
     4261 tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct
     4321 aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg
     4381 gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct
     4441 cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc
     4501 agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca gaataagttg
     4561 gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg
     4621 tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg
     4681 tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt
     4741 caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag
     4801 tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc
     4861 cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc
     4921 ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg
     4981 ggttcctggc tctgctcttc agactgagcc ccgttcccct gcatccccgt tcccctgcat
     5041 cccccttccc ctgcatcccc cagaggcccc aggccaccta cttggcctgg accccacgag
     5101 aggccacccc agccctgtct accaggctgc cttttgggtg gattctcctc caactgtggg
     5161 gtgactgctt ggcaaactca ctcttcgggg tatcccagga ggcctggagc attggggtgg
     5221 gctggggttc agagaggagg gattcccttc tcaggttacg tggccaagaa gcaggggagc
     5281 tgggtttggg tcaggtctgg gtgtggggtg accagcttat gctgtttgcc caggacagcc
     5341 tagttttagc actgaaaccc tcagtcctag gtgttcacca ggtcgtggcc gcctctactc
     5401 cctttctctt tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt
     5461 cctccgccca gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag
     5521 tccttggcaa gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc
     5581 tcatgcccct ggctctcctg ccccttccct acaggggttc ctggctctgc tctaagggcg
     5641 aattcgatct ggcgccatcg atacgcgtac gtcgcgaccg cggacatgta cagagctcga
     5701 gaagtactag tggccacgtg ggccgtgcac cttaagcttt taaataagga ggaataacat
     5761 atgaccatga ttacgccaag ctccaattcg ccctatagtg agtcgtatta caattcactg
     5821 gccgtcgttt tactatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     5881 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5941 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6001 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6061 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6121 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6181 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6241 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6301 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6361 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6421 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6481 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     6541 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6601 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6661 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6721 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6781 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6841 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6901 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6961 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7021 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7081 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7141 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7201 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7261 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7321 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7381 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7441 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7501 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7561 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7621 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7681 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7741 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7801 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     7861 aaaataggcg tatcacgagg c
//
