LOCUS       sequence-376091-        8747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knocking p2a-tdTomato into PTPRO locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8747)
  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 8747)
  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-PTPRO-LA-p2a-tdTomato-hGHpA-PGK-puroR-PT...
FEATURES             Location/Qualifiers
     source          1..8747
                     /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             3695..3751
                     /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             3752..5182
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    5247..5723
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5446..5465)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        5784..6283
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5819..5842)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6216..6235
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6323..6922
                     /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(6323..6342)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(8553..8572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8554..8572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8579..8596)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8579..8595)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8588..8610)
                     /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 accttggcct agtggttgat gtcctttact tatttgtcta tttgtatatg
     2701 ttactgattt gaatgacttt atcacaaaaa taaaggcatc aatattctgt cataattcta
     2761 gaaaatattt ttctttccca gtttgtggtt taccctgtgt tttattcttt ttaacttttt
     2821 aaaatataca gtagattgag atttttattt gtaatgtatt ctactgctct taattttaga
     2881 aattcctttc ctaaaggaat cagagagaaa ttaaccaata ttttcctctg tatttttaaa
     2941 gacaatttat taaattacct ggaatttgct ttggtgtatg ataatgggat atcctttttt
     3001 tttcaaaata gccccatgat ttgattgaat attcatttcc tttagcatta ataatgattt
     3061 aatgtacagc atggtgaaca tagttcataa taccgtattg tatacttgaa atttgctagg
     3121 aaagtagatc ttaagtgttc tcaccacaca cacacacaca caaatatata tacatacata
     3181 tatatgtata tatacacaca catatatata aacatatata taatctgggg tgatgaatgt
     3241 gttaactagc ttgattgtgg taatcatttc ataacatata tgtatatcaa gtcatcacat
     3301 tgtacacctt aaatatatac aatttttatt atactgcaat aaagttaggg gaaaaaaatt
     3361 ccactgtttt tttaaaaaag ccatgatgtt ctaagctctg ttattaatca aaatacttgt
     3421 acagttgatt ctcttggatt ttctagttat aaaactgtgt ctctgtaaat aatgaaaatg
     3481 gtttccttta acaccagatc ttgatcatta aaattgtcat agtataaaat attatatctt
     3541 tgcacatgtc tgctctgaaa cctgtttttg tcttttgttc caggagcagt acatttttat
     3601 ccatcagtgt gtgcaactga tgtggatgaa gaagaagcag cagttctgca tcagtgatgt
     3661 catatacgag aatgttagca agtccggatc cggcgcaaca aacttctctc tgctgaaaca
     3721 agccggagat gtcgaagaga atcctggacc gatggtgagc aagggcgagg aggtcatcaa
     3781 agagttcatg cgcttcaagg tgcgcatgga gggctccatg aacggccacg agttcgagat
     3841 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3901 caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc
     3961 caaggcgtac gtgaagcacc ccgccgacat ccccgattac aagaagctgt ccttccccga
     4021 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggtctggtga ccgtgaccca
     4081 ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt
     4141 cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg
     4201 cctgtacccc cgcgacggcg tgctgaaggg cgagatccac caggccctga agctgaagga
     4261 cggcggccac tacctggtgg agttcaagac catctacatg gccaagaagc ccgtgcaact
     4321 gcccggctac tactacgtgg acaccaagct ggacatcacc tcccacaacg aggactacac
     4381 catcgtggaa cagtacgagc gctccgaggg ccgccaccac ctgttcctgg ggcatggcac
     4441 cggcagcacc ggcagcggca gctccggcac cgcctcctcc gaggacaaca acatggccgt
     4501 catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg gccacgagtt
     4561 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     4621 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcatgta
     4681 cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga agctgtcctt
     4741 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt
     4801 gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga tgcgcggcac
     4861 caacttcccc cccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     4921 cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg ccctgaagct
     4981 gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca agaagcccgt
     5041 gcaactgccc ggctactact acgtggacac caagctggac atcacctccc acaacgagga
     5101 ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt tcctgtacgg
     5161 catggacgag ctgtacaagt aatgagaatt ctcaatttct gagaactgtc attctcggaa
     5221 attgaagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     5281 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     5341 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     5401 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     5461 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     5521 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     5581 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     5641 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     5701 ccactgctcc cttccctgtc ctttaggtct gaagaggagt ttacgtccag ccacgcgtag
     5761 gatctcgacc tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     5821 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     5881 acattccaca tccaccggta ggcgccaacc gactccgttc tttggtggcc ccttcgcgcc
     5941 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     6001 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     6061 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     6121 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     6181 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     6241 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga
     6301 tctcgagcag ctgaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     6361 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     6421 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     6481 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     6541 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     6601 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     6661 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     6721 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     6781 cgccggggtg cccgccttcc tggagacctc cgcgccccac aacctcccct tctacgagcg
     6841 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     6901 gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag
     6961 cgcacgaccc catgcatcga tgatatcaga tccccgggat gcagaaattg atgatctatt
     7021 aaacaataaa gatgtccact aaaatggaag tttttcctgt catactttgt taagaagggt
     7081 gagaacagag tacctacatt ttgaatggaa ggattggagc tacgggggtg ggggtggggt
     7141 gggattagat aaatgcctgc tctttactga aggctcttta ctattgcttt atgataatgt
     7201 ttcatagttg gatatcataa tttaaacaag caaaaccaaa ttaagggcca gctcattcct
     7261 cccactcatg atctatagat ctatagatct ctcgtgggat cattgttttt ctcttgattc
     7321 ccactttgtg gttctaagta ctgtggtttc caaatgtgtc agtttcatag cctgaagaac
     7381 gagatcagca gcctctgttc cacatacact tcattctcag tattgttttg ccaagttcta
     7441 attccatcag aagctggtcg agatccggaa cccttaattc aatttctgag aactgtcatt
     7501 ctcggaaatt gaggatccta gttcagaatc cggagcagta agtggagaag agctctccaa
     7561 tagtgctcag tcttagaact attagctggg gatgtagtgg atgggacaaa agacagcagt
     7621 agccatttgt attgactctg acttcacatg agtatttctt cccagctcct ggcctcacat
     7681 gggtggtcac agggagaaac aagtactgta gcagatcttt gagtttcaat gcaccttggt
     7741 tggtgttggc aacaaacagg agcttctgca catcatgatg actttacctc cattccctct
     7801 acccagaacc ctaaattatt ctgttattgg atctgtgcct gtaatatcag gatactacca
     7861 tccaaatcta tctagatatc ttgtgcaaat attttattga ttacaagggc tgtatacggg
     7921 gttacaaaaa gacatgagct ggattggaca ctcctaaaac caaaaacaac agtgacaata
     7981 gagtctcagg atagcagagt ctgaaaatac cttggtctcc cccattatgt gagcccttat
     8041 ttgagagctt catgaagaca cttgtaacat attcaacgtc ctgtactcca gttttgacta
     8101 cgagatcaga ttttggaggt gtaagtaatc ttggctgagg taacagtaat ctgcccattt
     8161 agttcagtaa acagactgta gagtgtctga ctatactggg ggaagaggag ccaatgacag
     8221 ggtagggaga tgacagtggt gcatgatggg agaatcatga aagtctggga agcatatgga
     8281 ccagtgggaa ggataggatt agcactgcac ctctacatcc atcaagagag acagagcgca
     8341 caatggagca ggaaggataa aaaataaaga aagacagaga caaacaaatt tgctgggaga
     8401 gggaaagaca ttcagaccca cagaaaggag gcagcacaca gagaaagatg cacagatcaa
     8461 cccacatctg catgggttgt gagactgagc atagtgagag cagtggtgga gcaatagaag
     8521 agcggccgcc accgcggtgg agctccaatt cgccctatag tgagtcgtat tacaattcac
     8581 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     8641 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     8701 cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcg
//
