LOCUS       sequence-376426-        8045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knocking p2a-EGFP into OTX2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8045)
  AUTHORS   Xu P, He H, Gao Q, Zhou Y, Wu Z, Zhang X, Sun L, Hu G, Guan Q, You 
            Z, Zhang X, Zheng W, Xiong M, Chen Y
  TITLE     Human midbrain dopaminergic neuronal differentiation markers predict
            cell therapy outcomes in a Parkinson's disease model.
  JOURNAL   J Clin Invest. 2022 Jul 15;132(14). pii: 156768. doi: 
            10.1172/JCI156768.
  PUBMED    35700056
REFERENCE   2  (bases 1 to 8045)
  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: "J Clin 
            Invest."; date: "2022-07-15"; volume: "132(14). pii: 156768. doi"; 
            pages: " 10.1172/JCI156768"
COMMENT     Sequence Label: PL752-OTX2-LA-p2a-EGFPnls-hGHpA-PGK-puroR-OTX2-RA
FEATURES             Location/Qualifiers
     source          1..8045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(16..444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(126..147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     338..357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        471..575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(576..644,645..1436)
                     /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     complement(794..813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1607..2195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2096..2115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2349..2366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2483..2504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2519..2524,2525..2542,2543..2549)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2557..2573
                     /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)."
     primer_bind     2562..2584
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2581..2597
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2581..2597
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2618..2636
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3670..3726
                     /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             join(3727..3729,3730..3732,3733..4443)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3727..3729,3730..3732,3733..4443)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3772..3793)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4033..4052)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4380..4401
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4444..4491
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    4559..5035
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4758..4777)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        5096..5583
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5131..5154)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5516..5535
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5623..6222
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5623..5642)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(7851..7870)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7852..7870)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7877..7894)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7877..7893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7886..7908)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
       61 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
      121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
      181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      421 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      541 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      601 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      661 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      721 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
      781 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
      841 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
      901 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
      961 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1021 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1081 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1141 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1201 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1261 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1321 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1381 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1441 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1501 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1561 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1621 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1681 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1741 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1801 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1861 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1921 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     1981 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2041 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2101 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2161 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2221 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2281 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2341 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2401 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2461 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     2521 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     2581 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac
     2641 aaaagctggt accgggcccc ccctcgaggc ctccaaaaga cagagataca ttctattcct
     2701 ctgtgagagt gttccaaaat atagctgggt ctatgtgtat gtgtgtgtta gaggcgaaat
     2761 aagataggaa agtgatgtgg agagttttta gctatatgat ttgggggatt atcttaaatg
     2821 ttattctggg caagaaataa gacagtgtca agtgttttta gacagagcct ccccaacttt
     2881 cttacaagtc caggagttta tatgagagta ccacataata ggtcttcagt ggcaggggaa
     2941 attgtgtgtt tagctgatct gcccatgtag gatagattta taatacggga gccattcttg
     3001 tccttaagga actatcaaaa ccgagttaaa gaattttctt tcccttccaa ggtatggttt
     3061 aagaatcgaa gagctaagtg ccgccaacaa cagcaacaac agcagaatgg aggtcaaaac
     3121 aaagtgagac ctgccaaaaa gaagacatct ccagctcggg aagtgagttc agagagtgga
     3181 acaagtggcc aattcactcc cccctctagc acctcagtcc cgaccattgc cagcagcagt
     3241 gctcctgtgt ctatctggag cccagcttcc atctccccac tgtcagatcc cttgtccacc
     3301 tcctcttcct gcatgcagag gtcctatccc atgacctata ctcaggcttc aggttatagt
     3361 caaggatatg ctggctcaac ttcctacttt gggggcatgg actgtggatc atatttgacc
     3421 cctatgcatc accagcttcc cggaccaggg gccacactca gtcccatggg taccaatgca
     3481 gtcaccagcc atctcaatca gtccccagct tctctttcca cccagggata tggagcttca
     3541 agcttgggtt ttaactcaac cactgattgc ttggattata aggaccaaac tgcctcctgg
     3601 aagcttaact tcaatgctga ctgcttggat tataaagatc agacatcctc gtggaaattc
     3661 caggttttgg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct
     3721 ggaccgatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     3781 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     3841 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3901 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     3961 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     4021 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     4081 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     4141 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     4201 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     4261 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4321 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     4381 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     4441 aagaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa ataatgagaa
     4501 ttctcaattt ctgagaactg tcattctcgg aaattgaagc gctgctcgag agatctacgg
     4561 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     4621 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     4681 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     4741 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     4801 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     4861 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4921 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4981 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tcctttaggt
     5041 ctgaagagga gtttacgtcc agccacgcgt aggatctcga cctcgaaatt ctaccgggta
     5101 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     5161 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     5221 ccgactccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     5281 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     5341 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     5401 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     5461 tccgggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     5521 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     5581 tcgacctgca tccatctaga tctcgagcag ctgaagctta ccatgaccga gtacaagccc
     5641 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     5701 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     5761 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     5821 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     5881 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     5941 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     6001 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     6061 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccac
     6121 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     6181 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
     6241 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga tgatatcaga tccccgggat
     6301 gcagaaattg atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt
     6361 catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc
     6421 tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta
     6481 ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa
     6541 ttaagggcca gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat
     6601 cattgttttt ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc
     6661 agtttcatag cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag
     6721 tattgttttg ccaagttcta attccatcag aagctggtcg agatccggaa cccttaattc
     6781 aatttctgag aactgtcatt ctcggaaatt gaggatcctg aagacctaga gaacctcttt
     6841 ttgtgggtga tttttaaata tactgggctg gacattccaa ccttagccag gcattggtta
     6901 aaagagttag atgggatgat gctcagactc atctgatcaa agttccgaga ggcatagaag
     6961 gaaaaacgaa gggccttaga ggggcctaca aaccagcaac atgaaatgga caaaccaatc
     7021 tgcttaagat cctgtcatag ttttagatca ttggttatcc tgatttgcaa agtgatcaaa
     7081 agcattctag ccatgtgcaa ccaaacacca ccaaaaataa aatcaaacaa aactaagttg
     7141 tgaaggaagg gagggaaggt catagccttc ttaagcagag gtgttccatt gttttagcca
     7201 atccttggtt gaatcttagg aatgaacagt gtctcaagct cattcacgtt tcatgaccaa
     7261 ctggtagttg gcactgaaaa aacttttcag ggctgtgtga attgtgtgac tgattgtcct
     7321 agatgcacta ctttatttaa aaaataatgt tcataaggag tcaatatgta gtttaagaga
     7381 caatcagtgt gtgtcttata aatggtacat ctgtggtttt taatctgtgc tagacttcaa
     7441 aactgtgatc tcctgttatt gtatgcaacc ttgaactcca cctctgcagg ggttcttctg
     7501 tgattaaata ggttataatt ataagcaaaa ttcagagcaa ctgagtactg atctaaaaag
     7561 attacctttg gctggaggtg agctgcactg aaactttacg acaaaatgtc tctggacaaa
     7621 gagagtcaga gaagagaagc aaaaggacac taattcatct gtaatttact gttggtaagc
     7681 ctagcagtaa agagacattg gtcaattgct ctgaccctga tgaattatta aactgagatc
     7741 attgtcgttt atgcttgcag atgttaaatg gaaaagttat atatgcataa accttttctt
     7801 cctggatttg gcagatatgg cggccgccac cgcggtggag ctccaattcg ccctatagtg
     7861 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     7921 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     7981 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
     8041 aagcg
//
