LOCUS       sequence-205803-        9307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TALEN (right) targeting HPRT exon3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9307)
  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 9307)
  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..9307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..24
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8..24
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    480..496
                     /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)."
     enhancer        523..902
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        904..1179
                     /label=chicken beta-actin promoter
     intron          1180..2188
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2112..2134
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2286..2305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2286..2304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           2312..2354
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      2362..2371
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2371..2436
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2443..2463
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4783..5370
                     /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(5388..5405)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5394..5505
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5623..5643)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5628..5957
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5808..5943
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5870..5889
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    7159..7280
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7196..7215)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7250..7269
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7333..8124,8125..8193))
                     /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     7956..7975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8194..8285)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8327..8346
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8352..8371
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8457..9045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8946..8965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9199..9216
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt ctgataacga
       61 gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt tatgggggga
      121 gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg cttgatatat
      181 gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa gatacgttgc
      241 tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg attttttgta
      301 tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa aataataaag
      361 ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa atataataca
      421 aactataaga tgttatcagt atttattatc atttagaata aattttgtgt cgcccttaat
      481 tgtgagcgga taacaattac gagcttcatg cacagtggcg ttgacattga ttattgacta
      541 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      601 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      661 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      721 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      781 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      841 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      901 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
      961 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
     1021 gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
     1081 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
     1141 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg
     1201 ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     1261 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
     1321 taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc
     1381 cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
     1441 cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1501 gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     1561 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1621 gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt gctgagcacg
     1681 gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg
     1741 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     1801 cgggggaggg gcgcggcggc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1861 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1921 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt
     1981 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     2041 cccttctcca tctccagcct cggggctgcc gcagggggac ggctgccttc gggggggacg
     2101 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     2161 tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc
     2221 tcatcatttt ggcaaagaat tctctggcta actagagaac ccactgctta ctggcttatc
     2281 gaaattaata cgactcacta tagggaagct tcttgttctt tttgcagaag ctcagaataa
     2341 acgctcaact ttggcctcga ggccaccatg gactataagg accacgacgg agactacaag
     2401 gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
     2461 gtcggtatcc acggagtccc agcagccgta gatttgagaa ctttgggata ttcacagcag
     2521 cagcaggaaa agatcaagcc caaagtgagg tcgacagtcg cgcagcatca cgaagcgctg
     2581 gtgggtcatg ggtttacaca tgcccacatc gtagccttgt cgcagcaccc tgcagccctt
     2641 ggcacggtcg ccgtcaagta ccaggacatg attgcggcgt tgccggaagc cacacatgag
     2701 gcgatcgtcg gtgtggggaa acagtggagc ggagcccgag cgcttgaggc cctgttgacg
     2761 gtcgcgggag agctgagagg gcctcccctt cagctggaca cgggccagtt gctgaagatc
     2821 gcgaagcggg gaggagtcac ggcggtcgag gcggtgcacg cgtggcgcaa tgcgctcacg
     2881 ggagcacccc tcaacctgac cccagaccag gttgtggcca tcgccagcaa cataggtggc
     2941 aagcaggccc tcgaaaccgt ccagagactg ttaccggttc tctgccagga ccacggcctg
     3001 acccccgaac aggttgtcgc tattgctagt aacggcggag gcaaacaggc gctggaaaca
     3061 gttcagcgcc tcttgccggt cttgtgtcag gcccacggcc tgaccccaga ccaggttgtg
     3121 gccatcgcca gcaacatagg tggcaagcag gccctcgaaa ccgtccagag actgttaccg
     3181 gttctctgcc aggcccacgg cctgaccccg gcccaggtgg ttgcaatcgc gtcacacgat
     3241 gggggaaagc aggccctaga aaccgttcag cgactcctgc ccgtcctgtg ccaggaccac
     3301 ggcctgaccc ccgaccaggt tgtcgctatt gctagtaacg gcggaggcaa acaggcgctg
     3361 gaaacagttc agcgcctctt gccggtcttg tgtcaggacc acggcctgac cccggaacag
     3421 gtggttgcaa tcgcgtcaca cgatggggga aagcaggccc tagaaaccgt tcagcgactc
     3481 ctgcccgtcc tgtgccaggc ccacggcctg accccagacc aggttgtggc catcgccagc
     3541 aacataggtg gcaagcaggc cctcgaaacc gtccagagac tgttaccggt tctctgccag
     3601 gcccacggcc tgaccccggc ccaggtggtt gcaatcgcgt cacacgatgg gggaaagcag
     3661 gccctagaaa ccgttcagcg actcctgccc gtcctgtgcc aggaccacgg cctgacccca
     3721 gaccaggttg tggccatcgc cagcaacata ggtggcaagc aggccctcga aaccgtccag
     3781 agactgttac cggttctctg ccaggaccac ggcctgaccc cggaacaggt ggttgcaatc
     3841 gcgtcacacg atgggggaaa gcaggcccta gaaaccgttc agcgactcct gcccgtcctg
     3901 tgccaggccc acggcctgac cccagaccag gttgtggcca tcgccagcaa cataggtggc
     3961 aagcaggccc tcgaaaccgt ccagagactg ttaccggttc tctgccaggc ccacggcctg
     4021 accccagccc aggttgtggc catcgccagc aacataggtg gcaagcaggc cctcgaaacc
     4081 gtccagagac tgttaccggt tctctgccag gaccacggcc tgacccccga ccaggttgtc
     4141 gctattgcta gtaacggcgg aggcaaacag gcgctggaaa cagttcagcg cctcttgccg
     4201 gtcttgtgtc aggaccacgg cctgacccca gaacaggttg tggccatcgc cagcaacata
     4261 ggtggcaagc aggccctcga aaccgtccag agactgttac cggttctctg ccaggcccac
     4321 ggcctgaccc cagaccaagt tgtcgcgatt gcaagcaaca acggaggcaa acaagcctta
     4381 gaaacagtcc agagattgtt gcctgtgctg tgccaagccc acggcctgac cccggcccag
     4441 gtggttgcaa tcgcgtcaca cgatggggga aagcaggccc tagaaaccgt tcagcgactc
     4501 ctgcccgtcc tgtgccagga ccacggcctg acccctgagc aggtagtggc tattgcatcc
     4561 aacggagggg gcagacccgc actggagtca atcgtggccc agctttcgag gccggacccc
     4621 gcgctggccg cactcactaa tgatcatctt gtagcgctgg cctgcctcgg cggacgaccc
     4681 gccttggatg cggtgaagaa ggggctcccg cacgcgcctg cattgattaa gcggaccaac
     4741 agaaggatcc ccgagaggac atcacatcga gtggcaggtt cccaactcgt gaagagtgaa
     4801 cttgaggaga aaaagtcgga gctgcggcac aaattgaaat acgtaccgca tgaatacatc
     4861 gaacttatcg aaattgctag gaactcgact caagacagaa tccttgagat gaaggtaatg
     4921 gagttcttta tgaaggttta tggataccga gggaagcatc tcggtggatc acgaaaaccc
     4981 gacggagcaa tctatacggt ggggagcccg attgattacg gagtgatcgt cgacacgaaa
     5041 gcctacagcg gtgggtacaa tcttcccatc gggcaggcag atgagatgca acgttatgtc
     5101 gaagaaaatc agaccaggaa caaacacatc aatccaaatg agtggtggaa agtgtatcct
     5161 tcatcagtga ccgagtttaa gtttttgttt gtctctgggc atttcaaagg caactataag
     5221 gcccagctca cacggttgaa tcacattacg aactgcaatg gtgcggtttt gtccgtagag
     5281 gaactgctca ttggtggaga aatgatcaaa gcgggaactc tgacactgga agaagtcaga
     5341 cgcaagttta acaatggcga gatcaatttc cgctcataaa aaatcagcct cgactgtgcc
     5401 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5461 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcacaac actcaaccct
     5521 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5581 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     5641 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5701 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5761 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     5821 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     5881 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     5941 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     6001 acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     6061 tcgacagcgt ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     6121 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     6181 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     6241 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     6301 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     6361 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     6421 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     6481 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     6541 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     6601 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     6661 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     6721 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     6781 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     6841 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     6901 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     6961 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     7021 aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     7081 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     7141 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     7201 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     7261 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     7321 taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7381 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7441 accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg ctccggattt
     7501 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7561 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7621 tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7681 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7741 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7801 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7861 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7921 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7981 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8041 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8101 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8161 aataagggcg acacggaaat gttgaatact catattcttc ctttttcaat attattgaag
     8221 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8281 acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg cgagcccatt
     8341 tatacctgaa tatggctcat aacacccctt gctcatgacc aaaatccctt aacgtgagtt
     8401 acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     8461 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     8521 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     8581 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca ccacttcaag
     8641 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     8701 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     8761 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     8821 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     8881 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     8941 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     9001 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     9061 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     9121 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     9181 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga gagtagggaa
     9241 ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc gtttctacaa
     9301 actcttt
//
