LOCUS       sequence-81843-e        5973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TALEN-mediated gene editing at human PPP1R12C/AAVS1 locus .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  AUTHORS   Luo Y, Liu C, Cerbini T, San H, Lin Y, Chen G, Rao MS, Zou J
  TITLE     Stable Enhanced Green Fluorescent Protein Expression After 
            Differentiation and Transplantation of Reporter Human Induced 
            Pluripotent Stem Cells Generated by AAVS1 Transcription 
            Activator-Like Effector Nucleases.
  JOURNAL   Stem Cells Transl Med. 2014 May 15. pii: sctm.2013-0212.
  PUBMED    24833591
REFERENCE   2  (bases 1 to 5973)
  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: "Stem Cells 
            Transl Med. 2014 May 15. pii: sctm.2013-0212."
COMMENT     Sequence Label: pZT-AAVS1-R1
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        36..415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        416..619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     570..590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     664..683
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        664..682
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             751..816
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             823..843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3160..3747
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     primer_bind     complement(3797..3814)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3803..4027
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             4200..4994
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4200..4994
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4254..4273)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4864..4883
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      5320..5908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5809..5828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
       61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      241 ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      601 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
      661 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagctgatc
      721 cactagtcca gtgtggtgga attcgccatg gactacaaag accatgacgg tgattataaa
      781 gatcatgaca tcgattacaa ggatgacgat gacaagatgg cccccaagaa gaagaggaag
      841 gtgggcatcc acggggtacc tatggtggac ttgaggacac tcggttattc gcaacagcaa
      901 caggagaaaa tcaagcctaa ggtcaggagc accgtcgcgc aacaccacga ggcgcttgtg
      961 gggcatggct tcactcatgc gcatattgtc gcgctttcac agcaccctgc ggcgcttggg
     1021 acggtggctg tcaaatacca agatatgatt gcggccctgc ccgaagccac gcacgaggca
     1081 attgtagggg tcggtaaaca gtggtcggga gcgcgagcac ttgaggcctt gctgactgtg
     1141 gcgggtgagc ttagggggcc tccgctccag ctcgacaccg ggcagctgct gaagatcgcg
     1201 aagagagggg gagtaacagc ggtagaggca gtgcacgcct ggcgcaatgc gctcaccggg
     1261 gcccccctga acctgacccc ggaccaagtg gtggctatcg ccagcaacgg tggcggcaag
     1321 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     1381 ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct cgaaacggtg
     1441 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     1501 atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1561 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     1621 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1681 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1921 ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1981 catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg caagcaagcg
     2041 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     2101 caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2161 ctgttgccgg tgctgtgcca ggaccatggc ctgactccgg accaagtggt ggctatcgcc
     2221 agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagccacga tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct cgaaacggtg
     2461 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2521 atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2581 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacggtggc
     2641 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2701 ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc ggaccaagtg
     2821 gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     2941 ggtggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc tgatccggcg
     3001 ttggccgcgt tgaccaacga ccatctggtg gcgttggcat gtcttggtgg acgtcccgcg
     3061 ctcgatgcag tcaaaaaggg tctgcctcat gctcccgcat tgatcaaaag aaccaaccgg
     3121 cggattcccg agagaacttc ccatcgagtc gcgggatccc aactagtcaa aagtgaactg
     3181 gaggagaaga aatctgaact tcgtcataaa ttgaaatatg tgcctcatga atatattgaa
     3241 ttaattgaaa ttgccagaaa ttccactcag gatagaattc ttgaaatgaa ggtaatggaa
     3301 ttttttatga aagtttatgg atatagaggt aaacatttgg gtggatcaag gaaaccggac
     3361 ggagcaattt atactgtcgg atctcctatt gattacggtg tgatcgtgga tactaaagct
     3421 tatagcggag gttataatct gccaattggc caagcagatg aaatgcaacg atatgtcgaa
     3481 gaaaatcaaa cacgaaacaa acatatcaac cctaatgaat ggtggaaagt ctatccatct
     3541 tctgtaacgg aatttaagtt tttatttgtg agtggtcact ttaaaggaaa ctacaaagct
     3601 cagcttacac gattaaatca tatcactaat tgtaatggag ctgttcttag tgtagaagag
     3661 cttttaattg gtggagaaat gattaaagcc ggcacattaa ccttagagga agtcagacgg
     3721 aaatttaata acggcgagat aaactttaga tcttgataac tcgagtctag agggcccgtt
     3781 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3841 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3901 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3961 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4021 tctatggctt ctactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg
     4081 cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc tcgccgccaa
     4141 ggatctgatg gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca
     4201 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4261 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4321 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4381 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4441 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4501 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4561 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4621 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4681 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4741 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4801 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4861 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4921 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4981 acgagttctt ctgaattatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     5041 atctgtgcgg tatttcacac cgcatacagg tggcactttt cggggaaatg tgcgcggaac
     5101 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5161 ctgataaatg cttcaataat agcacgtgct aaaacttcat ttttaattta aaaggatcta
     5221 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5461 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5941 gggcttttgc tggccttttg ctcacatgtt ctt
//
