LOCUS       sequence-205682-        7867 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses TALEN (left) targeting HPRT exon3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7867)
  AUTHORS   Kim SI, Matsumoto T, Kagawa H, Nakamura M, Hirohata R, Ueno A, 
            Ohishi M, Sakuma T, Soga T, Yamamoto T, Woltjen K
  TITLE     Microhomology-assisted scarless genome editing in human iPSCs.
  JOURNAL   Nat Commun. 2018 Mar 5;9(1):939. doi: 10.1038/s41467-018-03044-y.
  PUBMED    29507284
REFERENCE   2  (bases 1 to 7867)
  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: "2018-03-5"; volume: "9(1):939. doi"; pages: " 
            10.1038/s41467-018-03044-y"
COMMENT     Sequence Label: CAG-Avr-HPRT-LEFT
FEATURES             Location/Qualifiers
     source          1..7867
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     18..37
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        18..36
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 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      94..103
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             103..168
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             175..195
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2821..3408
                     /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(3426..3443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3432..3543
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3661..3681)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3666..3995
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3846..3981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3908..3927
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    5197..5318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5234..5253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5288..5307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5371..6162,6163..6231))
                     /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     5994..6013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6232..6323)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6365..6384
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6390..6409
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6495..7083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6984..7003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7237..7254
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7352..7369
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7353..7369
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7825..7841
                     /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)."
ORIGIN
        1 tactggctta tcgaaattaa tacgactcac tatagggaag cttcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcctc gaggccacca tggactataa ggaccacgac
      121 ggagactaca aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag
      181 aagaagcgga aggtcggtat ccacggagtc ccagcagccg tagatttgag aactttggga
      241 tattcacagc agcagcagga aaagatcaag cccaaagtga ggtcgacagt cgcgcagcat
      301 cacgaagcgc tggtgggtca tgggtttaca catgcccaca tcgtagcctt gtcgcagcac
      361 cctgcagccc ttggcacggt cgccgtcaag taccaggaca tgattgcggc gttgccggaa
      421 gccacacatg aggcgatcgt cggtgtgggg aaacagtgga gcggagcccg agcgcttgag
      481 gccctgttga cggtcgcggg agagctgaga gggcctcccc ttcagctgga cacgggccag
      541 ttgctgaaga tcgcgaagcg gggaggagtc acggcggtcg aggcggtgca cgcgtggcgc
      601 aatgcgctca cgggagcacc cctcaacctg accccggacc aggtggttgc aatcgcgtca
      661 cacgatgggg gaaagcaggc cctagaaacc gttcagcgac tcctgcccgt cctgtgccag
      721 gaccacggcc tgaccccgga acaggtggtt gcaatcgcgt cacacgatgg gggaaagcag
      781 gccctagaaa ccgttcagcg actcctgccc gtcctgtgcc aggcccacgg cctgacccca
      841 gaccaggttg tggccatcgc cagcaacata ggtggcaagc aggccctcga aaccgtccag
      901 agactgttac cggttctctg ccaggcccac ggcctgaccc ccgcccaggt tgtcgctatt
      961 gctagtaacg gcggaggcaa acaggcgctg gaaacagttc agcgcctctt gccggtcttg
     1021 tgtcaggacc acggcctgac ccccgaccag gttgtcgcta ttgctagtaa cggcggaggc
     1081 aaacaggcgc tggaaacagt tcagcgcctc ttgccggtct tgtgtcagga ccacggcctg
     1141 accccggaac aggtggttgc aatcgcgtca cacgatgggg gaaagcaggc cctagaaacc
     1201 gttcagcgac tcctgcccgt cctgtgccag gcccacggcc tgaccccgga ccaggtggtt
     1261 gcaatcgcgt cacacgatgg gggaaagcag gccctagaaa ccgttcagcg actcctgccc
     1321 gtcctgtgcc aggcccacgg cctgaccccc gcccaggttg tcgctattgc tagtaacggc
     1381 ggaggcaaac aggcgctgga aacagttcag cgcctcttgc cggtcttgtg tcaggaccac
     1441 ggcctgaccc cagaccaggt tgtggccatc gccagcaaca taggtggcaa gcaggccctc
     1501 gaaaccgtcc agagactgtt accggttctc tgccaggacc acggcctgac ccccgaacag
     1561 gttgtcgcta ttgctagtaa cggcggaggc aaacaggcgc tggaaacagt tcagcgcctc
     1621 ttgccggtct tgtgtcaggc ccacggcctg accccagacc aagttgtcgc gattgcaagc
     1681 aacaacggag gcaaacaagc cttagaaaca gtccagagat tgttgcctgt gctgtgccaa
     1741 gcccacggcc tgaccccagc ccaggttgtg gccatcgcca gcaacatagg tggcaagcag
     1801 gccctcgaaa ccgtccagag actgttaccg gttctctgcc aggaccacgg cctgaccccg
     1861 gaccaggtgg ttgcaatcgc gtcacacgat gggggaaagc aggccctaga aaccgttcag
     1921 cgactcctgc ccgtcctgtg ccaggaccac ggcctgaccc ccgaacaggt tgtcgctatt
     1981 gctagtaacg gcggaggcaa acaggcgctg gaaacagttc agcgcctctt gccggtcttg
     2041 tgtcaggccc acggcctgac cccagaccaa gttgtcgcga ttgcaagcaa caacggaggc
     2101 aaacaagcct tagaaacagt ccagagattg ttgcctgtgc tgtgccaagc ccacggcctg
     2161 acccccgccc aggttgtcgc tattgctagt aacggcggag gcaaacaggc gctggaaaca
     2221 gttcagcgcc tcttgccggt cttgtgtcag gaccacggcc tgaccccaga ccaggttgtg
     2281 gccatcgcca gcaacatagg tggcaagcag gccctcgaaa ccgtccagag actgttaccg
     2341 gttctctgcc aggaccacgg cctgacccca gaacaagttg tcgcgattgc aagcaacaac
     2401 ggaggcaaac aagccttaga aacagtccag agattgttgc cggtgctgtg ccaagcccac
     2461 ggcctgaccc cagaccaggt tgtggccatc gccagcaaca taggtggcaa gcaggccctc
     2521 gaaaccgtcc agagactgtt accggttctc tgccaggccc acggcctgac ccctgagcag
     2581 gtagtggcta ttgcatccaa cggagggggc agacccgcac tggagtcaat cgtggcccag
     2641 ctttcgaggc cggaccccgc gctggccgca ctcactaatg atcatcttgt agcgctggcc
     2701 tgcctcggcg gacgacccgc cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca
     2761 ttgattaagc ggaccaacag aaggatcccc gagaggacat cacatcgagt ggcaggttcc
     2821 caactcgtga agagtgaact tgaggagaaa aagtcggagc tgcggcacaa attgaaatac
     2881 gtaccgcatg aatacatcga acttatcgaa attgctagga actcgactca agacagaatc
     2941 cttgagatga aggtaatgga gttctttatg aaggtttatg gataccgagg gaagcatctc
     3001 ggtggatcac gaaaacccga cggagcaatc tatacggtgg ggagcccgat tgattacgga
     3061 gtgatcgtcg acacgaaagc ctacagcggt gggtacaatc ttcccatcgg gcaggcagat
     3121 gagatgcaac gttatgtcga agaaaatcag accaggaaca aacacatcaa tccaaatgag
     3181 tggtggaaag tgtatccttc atcagtgacc gagtttaagt ttttgtttgt ctctgggcat
     3241 ttcaaaggca actataaggc ccagctcaca cggttgaatc acattacgaa ctgcaatggt
     3301 gcggttttgt ccgtagagga actgctcatt ggtggagaaa tgatcaaagc gggaactctg
     3361 acactggaag aagtcagacg caagtttaac aatggcgaga tcaatttccg ctcataaaaa
     3421 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3481 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3541 atcacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3601 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3661 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3721 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3781 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3841 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3901 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3961 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4021 ttttcggatc tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga
     4081 agtttctgat cgaaaagttc gacagcgtcc gacctgatgc agctctcgga gggcgaagaa
     4141 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     4201 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     4261 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     4321 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     4381 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     4441 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     4501 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     4561 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     4621 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     4681 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     4741 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     4801 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     4861 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     4921 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     4981 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     5041 agcactcgtc cgagggcaaa ggaatagcac gtgctacgag atttcgattc caccgccgcc
     5101 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5161 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5221 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5281 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5341 tctagctaga gcttggcgta atcatggtca ttaccaatgc ttaatcagtg aggcacctat
     5401 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5461 tacgatacgg gagggcttac catctggccc cagcgctgcg atgataccgc gagaaccacg
     5521 ctcaccggct ccggatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5581 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5641 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt
     5701 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5761 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5821 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5881 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5941 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6001 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6061 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6121 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6181 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tattcttcct
     6241 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6301 atgtatttag aaaaataaac aaataggggt cagtgttaca accaattaac caattctgaa
     6361 cattatcgcg agcccattta tacctgaata tggctcataa caccccttgc tcatgaccaa
     6421 aatcccttaa cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     6481 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6541 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6601 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6661 ttagcccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6721 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6781 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6841 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6901 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6961 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7021 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7081 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7141 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7201 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7261 gaaggcgaga gtagggaact gccaggcatc aaactaagca gaaggcccct gacggatggc
     7321 ctttttgcgt ttctacaaac tctttctgtg ttgtaaaacg acggccagtc ttaagctcgg
     7381 gccccctggg cggttctgat aacgagtaat cgttaatccg caaataacgt aaaaacccgc
     7441 ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcaga atatttaagg
     7501 gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga gaaaaaagca
     7561 acgcacttta aataagatac gttgcttttt cgattgatga acacctataa ttaaactatt
     7621 catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa gagaattaag
     7681 aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa ttatacatgt
     7741 caacgataat acaaaatata atacaaacta taagatgtta tcagtattta ttatcattta
     7801 gaataaattt tgtgtcgccc ttaattgtga gcggataaca attacgagct tcatgcacag
     7861 tggcgtt
//
