LOCUS       sequence-376134-        8324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knocking p2a-mTagBFP2nls-3xHAtag into EN1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8324)
  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 8324)
  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-EN1-LA-p2a-mTagBFP2nls-3xHAtag-hGHpA-PGK-puroR-EN1-RA
FEATURES             Location/Qualifiers
     source          1..8324
                     /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             3783..3839
                     /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             4527..4574
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4578..4667
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    4735..5211
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4934..4953)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        5272..5771
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5307..5330)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5704..5723
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5811..6410
                     /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(5811..5830)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(8130..8149)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8131..8149)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8156..8173)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8156..8172)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8165..8187)
                     /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 ccctcgaggt cgacagccag ggtcatgagg atggatcagg
     2701 gccttttctc cttcagagaa ggggtggtcc ctgcatccct gagaccctag aaaaggcttc
     2761 ctttaggcca aagagaaaga aggcgaggag gtgtgtgtgg gagggagggg tgcggggagg
     2821 ggggcagaca ccaaatactt tggagctaag cagccctctc ccagccggct cactatcccg
     2881 ttgttttctg aggtcaatag ctacaagcag atggggtctt attgtagttt ctcctgtagg
     2941 tatgattctt gaggaggaga ggaagatttg tttgcaggct ggcgactccc ccagggcctg
     3001 ccctcactgc cccagtccta tctattccgg ggcggtgagg acagcaggcc cctgaccccc
     3061 aactctgggc tgggccatat gagctccaaa tatctcctca tcttggattc ctgccttctt
     3121 ggcttggaaa tgttgtgatt tcggtctgtc cctgactgtc ctgtgagaat tggggtgtgg
     3181 ggtgcaccta caacctcaac cagctttcct tcactgctcc tttgccctcc caaagggaac
     3241 ccggggaccc cagtggtctg gtgaggggca cttcgatcca gggattggct ggcgggaggg
     3301 agccaggggt tcagcagggc ctccgagatc gccccggaag cgcggggctc gcggcccagc
     3361 tcggcaatgc tattctcacg gcttcctttg ccctctcccc cctcccttct gccctcggtc
     3421 cccaccccca ccccaccccg gccctctccg ccgctgcagg tccgcgcacc aggaagctga
     3481 agaagaagaa gaacgagaag gaggacaagc ggccgcggac cgcgttcacg gccgagcagc
     3541 tgcagagact caaggcggag ttccaggcaa accgctacat cacggagcag cggcggcaga
     3601 ccctggccca ggaactcagc ctcaacgagt cccagatcaa gatctggttc cagaacaagc
     3661 gcgccaagat caagaaagcc acaggcatca agaacggcct ggcgctgcac ctcatggccc
     3721 agggactgta caaccactcc accaccacgg tccaggacaa agacgagagc gagggatccg
     3781 gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga
     3841 tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca
     3901 accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     3961 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta
     4021 gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca
     4081 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     4141 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     4201 tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     4261 aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg
     4321 ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca
     4381 agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa
     4441 gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat
     4501 actgcgacct ccctagcaaa ctggggaagc gacctgccgc cacaaagaag gctggacagg
     4561 ctaagaagaa gaaaggctac ccatacgatg ttccagatta cgctggctat ccctatgacg
     4621 tcccggacta tgcaggttca tatccatatg acgttccaga ttacgcttga taagaattct
     4681 caatttctga gaactgtcat tctcggaaat tgaagcgctg ctcgagagat ctacgggtgg
     4741 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     4801 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     4861 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     4921 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     4981 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     5041 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     5101 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     5161 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct ttaggtctga
     5221 agaggagttt acgtccagcc acgcgtagga tctcgacctc gaaattctac cgggtagggg
     5281 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     5341 gctacataag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccga
     5401 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     5461 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     5521 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     5581 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     5641 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     5701 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     5761 cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc atgaccgagt
     5821 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     5881 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     5941 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     6001 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     6061 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     6121 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     6181 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     6241 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     6301 cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     6361 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     6421 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg atatcagatc
     6481 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt
     6541 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg
     6601 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag
     6661 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca
     6721 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct
     6781 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca
     6841 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc
     6901 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc
     6961 cttaattcaa tttctgagaa ctgtcattct cggaaattga ggatcctagc cgccacaggc
     7021 cggggccgcg cccgcgcccc ctaacggcac cgccgccgtc gtctaacggc ccctcgctgg
     7081 gggagaaagc atctgctcca aggagggagg gagcgcaggg aaaagagcga gagagacaga
     7141 aagagagcct cagaatggac aatgacgttg aaacgcagca tttttgaaaa gggagaaaga
     7201 ctcggacagg tgctatcgaa aaataagatc cattctctat tcccagtata agggacgaaa
     7261 ctgcgaactc cttaaagctc tatctagcca aaccgcttac gaccttgtat atatttaatt
     7321 tcaggtaagg aaaacacata cgtgtagcga tctctatttg ctggacattt ttattaatct
     7381 cctttattat tattgttata attattataa ttattataat tattttatcc cctcccccac
     7441 cgcctcgctg cccccagccc agtttcgttt tcgttgcctt tttcatttga atgtcattgc
     7501 ttctccggtg cctcccgacc cgcatcgccg gccctggttt ctctgggact tttctttgtg
     7561 tgcgagagtg tgtttccttt cgtgtctgcc cacctcttct cccccacctc ccgggtccct
     7621 tctgtcggtc tgtctgttct gccccccttt cgttttccgg agacttgttg agaaatacga
     7681 ccccacagac tgcgagactg aaccgccgct acaagccaaa gattttatta tgttcagaaa
     7741 cctgtagtct gaaataaagt gtacactgtg ctcacgagcg ctggcggccc tcctgtttgc
     7801 gggctcggga aggacgtggg tccgggcaca cagcgcccca gcgcccctgc gcacgccggg
     7861 cggggagccg gccgggcctt gcgatcaccg cacaacgaat gcagggcgaa ggggaagcgc
     7921 gacccagatt tgggtttagc ctgggtctga agggtctggc gggacacgcc tggtctccga
     7981 ttgcgttcaa gggaactcaa aggccgacaa ccacagtcct ggcttcggaa acgctgcctg
     8041 ggctgcgggg cagctccccg caccggtcca ggcccagccc agtggcggcc tcctcctggc
     8101 ggccgccacc gcggtggagc tccaattcgc cctatagtga gtcgtattac aattcactgg
     8161 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     8221 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     8281 cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcg
//
