LOCUS       sequence-260082-        7608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knock in H2B-GFP into the human PAX6 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7608)
  AUTHORS   Tchieu J, Zimmer B, Fattahi F, Amin S, Zeltner N, Chen S, Studer L
  TITLE     A Modular Platform for Differentiation of Human PSCs into All Major 
            Ectodermal Lineages.
  JOURNAL   Cell Stem Cell. 2017 Sep 7;21(3):399-410.e7. doi: 
            10.1016/j.stem.2017.08.015.
  PUBMED    28886367
REFERENCE   2  (bases 1 to 7608)
  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"; doi: 
            "10.1016/j.stem.2017.08"; journalName: "Cell Stem Cell"; date: 
            "2017-09-7- 7"; volume: "21"; issue: "3"; pages: "399-410.e7"
COMMENT     Sequence Label: PAX6 TALEN R
FEATURES             Location/Qualifiers
     source          1..7608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      649..658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             658..723
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             730..750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2560..3147
                     /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(3165..3182)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3171..3282
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3400..3420)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3405..3734
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3585..3720
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3647..3666
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3783..4808
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3783..4808
                     /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    4938..5059
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4975..4994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5029..5048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5112..5903,5904..5972))
                     /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     5735..5754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5973..6064)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6106..6125
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6131..6150
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6236..6824
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6725..6744
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6978..6995
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7093..7110
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7094..7110
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7566..7582
                     /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 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctataggggc caccatggac
      661 tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat
      721 aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgtagat
      781 ttgagaactt tgggatattc acagcagcag caggaaaaga tcaagcccaa agtgaggtcg
      841 acagtcgcgc agcatcacga agcgctggtg ggtcatgggt ttacacatgc ccacatcgta
      901 gccttgtcgc agcaccctgc agcccttggc acggtcgccg tcaagtacca ggacatgatt
      961 gcggcgttgc cggaagccac acatgaggcg atcgtcggtg tggggaaaca gtggagcgga
     1021 gcccgagcgc ttgaggccct gttgacggtc gcgggagagc tgagagggcc tccccttcag
     1081 ctggacacgg gccagttgct gaagatcgcg aagcggggag gagtcacggc ggtcgaggcg
     1141 gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca acctgacccc agagcaggtc
     1201 gtggcaattg cgagcaacaa cgggggaaag caggcactcg aaaccgtcca gaggttgctg
     1261 cctgtgctgt gccaagcgca cggacttacg ccagagcagg tcgtggcaat tgcgagcaac
     1321 ggagggggaa agcaggcact cgaaaccgtc cagaggttgc tgcctgtgct gtgccaagcg
     1381 cacggactaa ccccagagca ggtcgtggca attgcgagca acatcggggg aaagcaggca
     1441 ctcgaaaccg tccagaggtt gctgcctgtg ctgtgccaag cgcacgggtt gaccccagag
     1501 caggtcgtgg caattgcgag caacggmggg ggaaagcagg cactcgaaac cgtccagagg
     1561 ttgctgcctg tgctgtgcca agcgcacggc ctgaccccag agcaggtcgt ggcaattgcg
     1621 agcaacawcg ggggaaagca ggcactcgaa accgtccaga ggttgctgcc tgtgctgtgc
     1681 caagcgcacg gactgacmcc agagcaggtc gtggcaattg cgagccatga cgggggaaag
     1741 caggcactcg aaaccgtcca gaggttgctg cctgtgctgt gccaagcgca cggacttaca
     1801 cccgaacaag tcgtggcaat tgcgagcaac atcgggggaa agcaggcact cgaaaccgtc
     1861 cagaggttgc tgcctgtgct gtgccaagcg cacggactta cgccagagca ggtcgtggca
     1921 attgcgagca acawcggggg aaagcaggca ctcgaaaccg tccagaggtt gctgcctgtg
     1981 ctgtgccaag cgcacggact raccccagag caggtcgtgg caattgcgag caacggmggg
     2041 ggaaagcagg cactcgaaac cgtccagagg ttgctgcctg tgctgtgcca agcgcacggc
     2101 ctgaccccag agcaggtcgt ggcaattgcg agcaacaacg ggggaaagca ggcactcgaa
     2161 accgtccaga ggttgctgcc tgtgctgtgc caagcgcacg gactgacacc agagcaggtc
     2221 gtggcaattg cgagcaacgg agggggaaag caggcactcg aaaccgtcca gaggttgctg
     2281 cctgtgctgt gccaagcgca cggactcacg cctgagcagg tagtggctat tgcatccaac
     2341 ggagggggca gacccgcact ggagtcaatc gtggcccagc tttcgaggcc ggaccccgcg
     2401 ctggccgcac tcactaatga tcatcttgta gcgctggcct gcctcggcgg acgacccgcc
     2461 ttggatgcgg tgaagaaggg gctcccgcac gcgcctgcat tgattaagcg gaccaacaga
     2521 aggattcccg agaggacatc acatcgagtg gcaggttccc aactcgtgaa gagtgaactt
     2581 gaggagaaaa agtcggagct gcggcacaaa ttgaaatacg taccgcatga atacatcgaa
     2641 cttatcgaaa ttgctaggaa ctcgactcaa gacagaatcc ttgagatgaa ggtaatggag
     2701 ttctttatga aggtttatgg ataccgaggg aagcatctcg gtggatcacg aaaacccgac
     2761 ggagcaatct atacggtggg gagcccgatt gattacggag tgatcgtcga cacgaaagcc
     2821 tacagcggtg ggtacaatct tcccatcggg caggcagatg agatgcaacg ttatgtcgaa
     2881 gaaaatcaga ccaggaacaa acacatcaat ccaaatgagt ggtggaaagt gtatccttca
     2941 tcagtgaccg agtttaagtt tttgtttgtc tctgggcatt tcaaaggcaa ctataaggcc
     3001 cagctcacac ggttgaatca cattacgaac tgcaatggtg cggttttgtc cgtagaggaa
     3061 ctgctcattg gtggagaaat gatcaaagcg ggaactctga cactggaaga agtcagacgc
     3121 aagtttaaca atggcgagat caatttccgc tcataaaaaa tcagcctcga ctgtgccttc
     3181 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3241 cactcccact gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc
     3301 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3361 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3421 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3481 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3541 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3601 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3661 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3721 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     3781 tgatgaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     3841 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     3901 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     3961 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4021 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4081 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4141 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     4201 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     4261 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     4321 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     4381 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     4441 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     4501 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     4561 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     4621 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     4681 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     4741 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     4801 aggaatagca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4861 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4921 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4981 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5041 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5101 aatcatggtc attaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5161 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5221 ccatctggcc ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta
     5281 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5341 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5401 agtttgcgca acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5461 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5521 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5581 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5641 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5701 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5761 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5821 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5881 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5941 ataagggcga cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc
     6001 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6061 caaatagggg tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt
     6121 atacctgaat atggctcata acaccccttg ctcatgacca aaatccctta acgtgagtta
     6181 cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6241 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6301 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6361 gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga
     6421 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6481 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6541 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6601 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6661 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6721 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6781 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6841 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6901 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6961 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac
     7021 tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa
     7081 ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga
     7141 taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg
     7201 gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg
     7261 atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata
     7321 cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta tttatgattt
     7381 tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata
     7441 ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat
     7501 aatacaaact ataagatgtt atcagtattt attatcattt agaataaatt ttgtgtcgcc
     7561 cttaattgtg agcggataac aattacgagc ttcatgcaca gtggcgtt
//
