LOCUS       sequence-211546-        7156 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 7156)
  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 7156)
  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 donor plasmid
FEATURES             Location/Qualifiers
     source          1..7156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1031..1087
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1088..1465
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1478..1487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1484..1486,1487..1489,1490..2203)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1484..1486,1487..1489,1490..2200)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1529..1550)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1790..1809)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2137..2158
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2216..2249)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2476..2986
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2905..2923
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3008..3607
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3008..3027)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3504..3524
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3614..3838
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3876..3909)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4557..4573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4557..4573)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4570..4592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4581..4597
                     /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)."
     promoter        complement(join(4605..4611,4612..4629,4630..4635))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4650..4671
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4788..4805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4959..5547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5039..5058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5718..6509,6510..6578))
                     /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     6341..6360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6579..6683)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6751..6769
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6807..6829)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6929..6948
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(7142..7156,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(7156,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg atcgtggctg tgtgatgtgt
       61 tcctcagtaa ccacaggttt gcctctctcc tcacagcccc cagtccccag ccagacctcc
      121 tcatactcct gcatgctgcc caccagccct tcggtgaatg ggcggagtta tgatacctac
      181 acccccccac atatgcagac acacatgaac agtcagccaa tgggcacctc gggcaccact
      241 tcaacaggtg agccactgct ttctgcaggc tgcacagagg cgatctctcg tcactagaag
      301 tttacccaaa cagaatctcc tggtcttatg ggagggcgtg tttaactcct tgctttcctt
      361 gtccctgggg gatggggatt gaaaagggaa attcagttaa gctaattagt aactttacac
      421 catatagaca aaaactaaaa ttgtttttcc tgaatttggt cacaaaagtt gtgtatgaag
      481 acaaggcctg agactgcaag ttttctgagg acagattatt agacgaagct cagtaggggg
      541 cccactgagc tgtaggtgcg tgcttgttga aatgcttctt gccctcatag ctcctctaga
      601 ccttttgctg gaaataaaaa gtgacacatt ggttttccag agacagcttt attgtaaaag
      661 ttccaaacat gcaaacaaac agaggatttt tttttctttt cctttggatt ggggtggggg
      721 gtacttggga tccaataggt atatatacat atattgtcta gtttctgaag gtgctacttt
      781 tatttgtaac aattgaagtg attttaatac agtaaaaaat gttagaaagt attagttttt
      841 tttttttttt tttttttttt gtaaacctat aaatttgtat tccatgtctg tttctcaaag
      901 ggaatatcta catggctatt tctttcatcc acttctagga ctcatttccc ctggtgtgtc
      961 agttccagtt caagttcccg gaagtgaacc tgatatgtct caatactggc caagattaca
     1021 gggcagcggc gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aagagaaccc
     1081 tggccccatg ccagagccag cgaagtctgc tcccgccccg aaaaagggct ccaagaaggc
     1141 ggtgactaag gcgcagaaga aaggcggcaa gaagcgcaag cgcagccgca aggagagcta
     1201 ttccatctat gtgtacaagg ttctgaagca ggtccaccct gacaccggca tttcgtccaa
     1261 ggccatgggc atcatgaatt cgtttgtgaa cgacattttc gagcgcatcg caggtgaggc
     1321 ttcccgcctg gcgcattaca acaagcgctc gaccatcacc tccagggaga tccagacggc
     1381 cgtgcgcctg ctgctgcctg gggagttggc caagcacgcc gtgtccgagg gtactaaggc
     1441 catcaccaag tacaccagcg ctaaggatcc accggtcgcc accatggtga gcaagggcga
     1501 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1561 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1621 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1681 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1741 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1801 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1861 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1921 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1981 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2041 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     2101 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     2161 cgccgccggg atcactctcg gcatggacga gctgtacaag taagctagcg gatccataac
     2221 ttcgtatagc atacattata cgaagttatg tgctagctct tccagccccc tgtcatggca
     2281 tcttccaggg gtccgagagc tcagctagtc ttcttcctcc aacccgggcc cctatgtcca
     2341 cttcaggaca gcatgtttgc tgcctccagg gatcctgtgt ccccgagctg ggaccacctt
     2401 atattcccag ggccggttaa tgtggctctg gttctgggta cttttatctg tcccctccac
     2461 cccacagtgg ggcaagcttt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca
     2521 gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg
     2581 cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc
     2641 cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg
     2701 ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca
     2761 gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg
     2821 gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt
     2881 gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc
     2941 ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga
     3001 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3061 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3121 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3181 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3241 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3301 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3361 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     3421 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3481 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3541 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3601 tgcctgaggt accctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3661 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3721 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3781 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggg
     3841 taccaagctt tactagggac aggattggtg acagaataac ttcgtatagc atacattata
     3901 cgaagttatt caacaagctt tgccacgaat ttaagagttt tatcaagata tatcgaatac
     3961 ttctacccat ctgttcatag tttatggact gatgttccaa gtttgtatca ttcctttgca
     4021 tataattaaa cctggaacaa catgcactag atttatgtca gaaatatctg ttggttttcc
     4081 aaaggttgtt aacagatgaa gtttatgtgc aaaaaagggt aagatataaa ttcaaggaag
     4141 aaaaaaagtt gatagctaaa aggtagagtg tgtcttcgat ataatccaat ttgttttatg
     4201 tcaaaatgta agtatttgtc ttccctagaa atcctcagaa tgatttctat aataaagtta
     4261 atttcattta tatttgacaa gaatatagat gttttataca cattttcatg caatcatacg
     4321 tttctttttt ggccagcaaa agttaattgt tcttagatat agttgtatta ctgttcacgg
     4381 tccaatcatt ttgtgcatct agagttcatt cctaatcaat taaaagtgct tgcaagagtt
     4441 ttaaacttaa gtgttttgaa gttgttcaca actacatatc aaaattaacc attgttgatt
     4501 gtaaaaaacg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca
     4561 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4621 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4681 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4741 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4801 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4861 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4921 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4981 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5041 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5101 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5161 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5221 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5281 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5341 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5401 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5461 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5521 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5581 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5641 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5701 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5761 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5821 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5881 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5941 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6001 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6061 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6121 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6181 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6241 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6301 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6361 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6421 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6481 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6541 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6601 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6661 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6721 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     6781 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6841 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6901 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     6961 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat
     7021 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     7081 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     7141 tcccagtcac gacgtt
//
