LOCUS       sequence-205802-        7552 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses TALEN (right) targeting HPRT exon3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7552)
  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 7552)
  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-RIGHT
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     16..35
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        16..34
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           42..84
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      92..101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             101..166
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             173..193
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2513..3100
                     /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(3118..3135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3124..3235
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3353..3373)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3358..3687
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3538..3673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3600..3619
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4889..5010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4926..4945)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4980..4999
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5063..5854,5855..5923))
                     /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     5686..5705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5924..6015)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6057..6076
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6082..6101
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6187..6775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6676..6695
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6929..6946
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7044..7061
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7045..7061
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7517..7533
                     /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 ctggcttatc gaaattaata cgactcacta tagggaagct tcttgttctt tttgcagaag
       61 ctcagaataa acgctcaact ttggcctcga ggccaccatg gactataagg accacgacgg
      121 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
      181 gaagcggaag gtcggtatcc acggagtccc agcagccgta gatttgagaa ctttgggata
      241 ttcacagcag cagcaggaaa agatcaagcc caaagtgagg tcgacagtcg cgcagcatca
      301 cgaagcgctg gtgggtcatg ggtttacaca tgcccacatc gtagccttgt cgcagcaccc
      361 tgcagccctt ggcacggtcg ccgtcaagta ccaggacatg attgcggcgt tgccggaagc
      421 cacacatgag gcgatcgtcg gtgtggggaa acagtggagc ggagcccgag cgcttgaggc
      481 cctgttgacg gtcgcgggag agctgagagg gcctcccctt cagctggaca cgggccagtt
      541 gctgaagatc gcgaagcggg gaggagtcac ggcggtcgag gcggtgcacg cgtggcgcaa
      601 tgcgctcacg ggagcacccc tcaacctgac cccagaccag gttgtggcca tcgccagcaa
      661 cataggtggc aagcaggccc tcgaaaccgt ccagagactg ttaccggttc tctgccagga
      721 ccacggcctg acccccgaac aggttgtcgc tattgctagt aacggcggag gcaaacaggc
      781 gctggaaaca gttcagcgcc tcttgccggt cttgtgtcag gcccacggcc tgaccccaga
      841 ccaggttgtg gccatcgcca gcaacatagg tggcaagcag gccctcgaaa ccgtccagag
      901 actgttaccg gttctctgcc aggcccacgg cctgaccccg gcccaggtgg ttgcaatcgc
      961 gtcacacgat gggggaaagc aggccctaga aaccgttcag cgactcctgc ccgtcctgtg
     1021 ccaggaccac ggcctgaccc ccgaccaggt tgtcgctatt gctagtaacg gcggaggcaa
     1081 acaggcgctg gaaacagttc agcgcctctt gccggtcttg tgtcaggacc acggcctgac
     1141 cccggaacag gtggttgcaa tcgcgtcaca cgatggggga aagcaggccc tagaaaccgt
     1201 tcagcgactc ctgcccgtcc tgtgccaggc ccacggcctg accccagacc aggttgtggc
     1261 catcgccagc aacataggtg gcaagcaggc cctcgaaacc gtccagagac tgttaccggt
     1321 tctctgccag gcccacggcc tgaccccggc ccaggtggtt gcaatcgcgt cacacgatgg
     1381 gggaaagcag gccctagaaa ccgttcagcg actcctgccc gtcctgtgcc aggaccacgg
     1441 cctgacccca gaccaggttg tggccatcgc cagcaacata ggtggcaagc aggccctcga
     1501 aaccgtccag agactgttac cggttctctg ccaggaccac ggcctgaccc cggaacaggt
     1561 ggttgcaatc gcgtcacacg atgggggaaa gcaggcccta gaaaccgttc agcgactcct
     1621 gcccgtcctg tgccaggccc acggcctgac cccagaccag gttgtggcca tcgccagcaa
     1681 cataggtggc aagcaggccc tcgaaaccgt ccagagactg ttaccggttc tctgccaggc
     1741 ccacggcctg accccagccc aggttgtggc catcgccagc aacataggtg gcaagcaggc
     1801 cctcgaaacc gtccagagac tgttaccggt tctctgccag gaccacggcc tgacccccga
     1861 ccaggttgtc gctattgcta gtaacggcgg aggcaaacag gcgctggaaa cagttcagcg
     1921 cctcttgccg gtcttgtgtc aggaccacgg cctgacccca gaacaggttg tggccatcgc
     1981 cagcaacata ggtggcaagc aggccctcga aaccgtccag agactgttac cggttctctg
     2041 ccaggcccac ggcctgaccc cagaccaagt tgtcgcgatt gcaagcaaca acggaggcaa
     2101 acaagcctta gaaacagtcc agagattgtt gcctgtgctg tgccaagccc acggcctgac
     2161 cccggcccag gtggttgcaa tcgcgtcaca cgatggggga aagcaggccc tagaaaccgt
     2221 tcagcgactc ctgcccgtcc tgtgccagga ccacggcctg acccctgagc aggtagtggc
     2281 tattgcatcc aacggagggg gcagacccgc actggagtca atcgtggccc agctttcgag
     2341 gccggacccc gcgctggccg cactcactaa tgatcatctt gtagcgctgg cctgcctcgg
     2401 cggacgaccc gccttggatg cggtgaagaa ggggctcccg cacgcgcctg cattgattaa
     2461 gcggaccaac agaaggatcc ccgagaggac atcacatcga gtggcaggtt cccaactcgt
     2521 gaagagtgaa cttgaggaga aaaagtcgga gctgcggcac aaattgaaat acgtaccgca
     2581 tgaatacatc gaacttatcg aaattgctag gaactcgact caagacagaa tccttgagat
     2641 gaaggtaatg gagttcttta tgaaggttta tggataccga gggaagcatc tcggtggatc
     2701 acgaaaaccc gacggagcaa tctatacggt ggggagcccg attgattacg gagtgatcgt
     2761 cgacacgaaa gcctacagcg gtgggtacaa tcttcccatc gggcaggcag atgagatgca
     2821 acgttatgtc gaagaaaatc agaccaggaa caaacacatc aatccaaatg agtggtggaa
     2881 agtgtatcct tcatcagtga ccgagtttaa gtttttgttt gtctctgggc atttcaaagg
     2941 caactataag gcccagctca cacggttgaa tcacattacg aactgcaatg gtgcggtttt
     3001 gtccgtagag gaactgctca ttggtggaga aatgatcaaa gcgggaactc tgacactgga
     3061 agaagtcaga cgcaagttta acaatggcga gatcaatttc cgctcataaa aaatcagcct
     3121 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3181 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcacaac
     3241 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3301 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3361 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3421 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3481 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3541 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3601 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3661 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3721 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3781 atcgaaaagt tcgacagcgt ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3841 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     3901 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     3961 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     4021 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     4081 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     4141 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     4201 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     4261 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     4321 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     4381 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4441 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4501 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4561 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4621 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     4681 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4741 tccgagggca aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga
     4801 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4861 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4921 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4981 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5041 gagcttggcg taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga
     5101 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5161 gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg
     5221 ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5281 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5341 cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct
     5401 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5461 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5521 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5581 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5641 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5701 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5761 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5821 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5881 caaaaaaggg aataagggcg acacggaaat gttgaatact catattcttc ctttttcaat
     5941 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6001 agaaaaataa acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg
     6061 cgagcccatt tatacctgaa tatggctcat aacacccctt gctcatgacc aaaatccctt
     6121 aacgtgagtt acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6181 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6241 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6301 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca
     6361 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6421 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6481 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6541 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6601 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6661 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6721 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6781 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6841 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6901 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga
     6961 gagtagggaa ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc
     7021 gtttctacaa actctttctg tgttgtaaaa cgacggccag tcttaagctc gggccccctg
     7081 ggcggttctg ataacgagta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg
     7141 gtttttttat ggggggagtt tagggaaaga gcatttgtca gaatatttaa gggcgcctgt
     7201 cactttgctt gatatatgag aattatttaa ccttataaat gagaaaaaag caacgcactt
     7261 taaataagat acgttgcttt ttcgattgat gaacacctat aattaaacta ttcatctatt
     7321 atttatgatt ttttgtatat acaatatttc tagtttgtta aagagaatta agaaaataaa
     7381 tctcgaaaat aataaaggga aaatcagttt ttgatatcaa aattatacat gtcaacgata
     7441 atacaaaata taatacaaac tataagatgt tatcagtatt tattatcatt tagaataaat
     7501 tttgtgtcgc ccttaattgt gagcggataa caattacgag cttcatgcac ag
//
