LOCUS       sequence-272442-        8118 bp    DNA     circular SYN 14-APR-2025
DEFINITION  TALEN designed to target the human AAVS1/PPP1R12C locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8118)
  AUTHORS   Gonzalez F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D
  TITLE     An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome 
            Editing in Human Pluripotent Stem Cells.
  JOURNAL   Cell Stem Cell. 2014 Jun 11. pii: S1934-5909(14)00205-7. doi: 
            10.1016/j.stem.2014.05.018.
  PUBMED    24931489
REFERENCE   2  (bases 1 to 8118)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem 
            Cell. 2014 Jun 11. pii: S1934-5909(14)00205-7. doi: 
            10.1016/j.stem.2014.05.018."
COMMENT     Sequence Label: AAVS1-TALEN-R
FEATURES             Location/Qualifiers
     source          1..8118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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        1459..1838
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1839..2042
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1993..2013
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2087..2106
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2087..2105
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2107..2116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2116..2181
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2188..2208
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4528..5115
                     /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(5133..5150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5139..5250
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5368..5388)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5373..5702
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5553..5688
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5615..5634
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5751..6776
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             5751..6776
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    6906..7027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6943..6962)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6997..7016
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7080..7871,7872..7940))
                     /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     7703..7722
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7941..8032)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8074..8093
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8099..8118
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1501 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1561 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1621 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1681 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1741 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1801 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1861 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1921 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1981 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     2041 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     2101 ataggggcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
     2161 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     2221 gtcccagcag ccgtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc
     2281 aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt
     2341 acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc
     2401 aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg
     2461 gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc gggagagctg
     2521 agagggcctc cccttcagct ggacacgggc cagttgctga agatcgcgaa gcggggagga
     2581 gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac
     2641 ctgaccccag agcaggtcgt ggcaattgcg agcaacggag ggggaaagca ggcactcgaa
     2701 accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg gacttacgcc agagcaggtc
     2761 gtggcaattg cgagcaacgg agggggaaag caggcactcg aaaccgtcca gaggttgctg
     2821 cctgtgctgt gccaagcgca cggactaacc ccagagcagg tcgtggcaat tgcgagcaac
     2881 ggagggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg
     2941 cacgggttga ccccagagca ggtcgtggca attgcgagcc atgacggggg aaagcaggca
     3001 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacggcct gaccccagag
     3061 caggtcgtgg caattgcgag caacggaggg ggaaagcagg cactcgaaac cgtccagagg
     3121 ttgctgcctg tgctgtgcca agcgcacgga ctgacaccag agcaggtcgt ggcaattgcg
     3181 agcaacaacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc
     3241 caagcgcacg gacttacacc cgaacaagtc gtggcaattg cgagcaacgg agggggaaag
     3301 caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca cggacttacg
     3361 ccagagcagg tcgtggcaat tgcgagccat gacgggggaa agcaggcact cgaaaccgtc
     3421 cagaggttgc tgcctgtgct gtgccaagcg cacggactaa ccccagagca ggtcgtggca
     3481 attgcgagca acatcggggg aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg
     3541 ctgtgccaag cgcacgggtt gaccccagag caggtcgtgg caattgcgag ccatgacggg
     3601 ggaaagcagg cactcgaaac cgtccagagg ttgctgcctg tgctgtgcca agcgcacggc
     3661 ctgaccccag agcaggtcgt ggcaattgcg agccatgacg ggggaaagca ggcactcgaa
     3721 accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg gactgacacc agagcaggtc
     3781 gtggcaattg cgagcaacat cgggggaaag caggcactcg aaaccgtcca gaggttgctg
     3841 cctgtgctgt gccaagcgca cggcctcacc ccagagcagg tcgtggcaat tgcgagcaac
     3901 atcgggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg
     3961 cacggactta cgccagagca ggtcgtggca attgcgagca acggaggggg aaagcaggca
     4021 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacggact aaccccagag
     4081 caggtcgtgg caattgcgag ccatgacggg ggaaagcagg cactcgaaac cgtccagagg
     4141 ttgctgcctg tgctgtgcca agcgcacggg ttgaccccag agcaggtcgt ggcaattgcg
     4201 agccatgacg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc
     4261 caagcgcacg gactcacgcc tgagcaggta gtggctattg catccaacgg agggggcaga
     4321 cccgcactgg agtcaatcgt ggcccagctt tcgaggccgg accccgcgct ggccgcactc
     4381 actaatgatc atcttgtagc gctggcctgc ctcggcggac gacccgcctt ggatgcggtg
     4441 aagaaggggc tcccgcacgc gcctgcattg attaagcgga ccaacagaag gattcccgag
     4501 aggacatcac atcgagtggc aggttcccaa ctcgtgaaga gtgaacttga ggagaaaaag
     4561 tcggagctgc ggcacaaatt gaaatacgta ccgcatgaat acatcgaact tatcgaaatt
     4621 gctaggaact cgactcaaga cagaatcctt gagatgaagg taatggagtt ctttatgaag
     4681 gtttatggat accgagggaa gcatctcggt ggatcacgaa aacccgacgg agcaatctat
     4741 acggtgggga gcccgattga ttacggagtg atcgtcgaca cgaaagccta cagcggtggg
     4801 tacaatcttc ccatcgggca ggcagatgag atgcaacgtt atgtcgaaga aaatcagacc
     4861 aggaacaaac acatcaatcc aaatgagtgg tggaaagtgt atccttcatc agtgaccgag
     4921 tttaagtttt tgtttgtctc tgggcatttc aaaggcaact ataaggccca gctcacacgg
     4981 ttgaatcaca ttacgaactg caatggtgcg gttttgtccg tagaggaact gctcattggt
     5041 ggagaaatga tcaaagcggg aactctgaca ctggaagaag tcagacgcaa gtttaacaat
     5101 ggcgagatca atttccgctc ataaaaaatc agcctcgact gtgccttcta gttgccagcc
     5161 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5221 cctttcctaa taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct
     5281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5341 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5401 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5461 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5521 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5581 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5641 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5701 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg atgaaaaagc
     5761 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     5821 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     5881 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     5941 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     6001 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     6061 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     6121 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     6181 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     6241 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     6301 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     6361 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     6421 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     6481 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     6541 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     6601 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     6661 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     6721 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg
     6781 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     6841 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     6901 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     6961 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     7021 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     7081 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7141 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7201 agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc agcaataaac
     7261 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7321 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7381 gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7441 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7501 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7561 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7621 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7681 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7741 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7801 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7861 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7921 cggaaatgtt gaatactcat attcttcctt tttcaatatt attgaagcat ttatcagggt
     7981 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtc
     8041 agtgttacaa ccaattaacc aattctgaac attatcgcga gcccatttat acctgaatat
     8101 ggctcataac accccttg
//
