LOCUS       sequence-376449-        8226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For knocking p2a-mNeonGreen into EN1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8226)
  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 8226)
  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: PL452-EN1-LA-p2a-mNeonGreen-hGHpA-PGK-neoR-EN1-RA
FEATURES             Location/Qualifiers
     source          1..8226
                     /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             3762..3818
                     /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"
     protein_bind    4582..4615
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    4637..5113
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4836..4855)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        5174..5673
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5209..5232)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5606..5625
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5685..5732
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5751..6554
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5814..5833)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6424..6443
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6586..6603)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6592..6816
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6873..6906
                     /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(8032..8051)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8033..8051)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8058..8075)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8058..8074)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8067..8089)
                     /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 accagccagg gtcatgagga tggatcaggg ccttttctcc ttcagagaag
     2701 gggtggtccc tgcatccctg agaccctaga aaaggcttcc tttaggccaa agagaaagaa
     2761 ggcgaggagg tgtgtgtggg agggaggggt gcggggaggg gggcagacac caaatacttt
     2821 ggagctaagc agccctctcc cagccggctc actatcccgt tgttttctga ggtcaatagc
     2881 tacaagcaga tggggtctta ttgtagtttc tcctgtaggt atgattcttg aggaggagag
     2941 gaagatttgt ttgcaggctg gcgactcccc cagggcctgc cctcactgcc ccagtcctat
     3001 ctattccggg gcggtgagga cagcaggccc ctgaccccca actctgggct gggccatatg
     3061 agctccaaat atctcctcat cttggattcc tgccttcttg gcttggaaat gttgtgattt
     3121 cggtctgtcc ctgactgtcc tgtgagaatt ggggtgtggg gtgcacctac aacctcaacc
     3181 agctttcctt cactgctcct ttgccctccc aaagggaacc cggggacccc agtggtctgg
     3241 tgaggggcac ttcgatccag ggattggctg gcgggaggga gccaggggtt cagcagggcc
     3301 tccgagatcg ccccggaagc gcggggctcg cggcccagct cggcaatgct attctcacgg
     3361 cttcctttgc cctctccccc ctcccttctg ccctcggtcc ccacccccac cccaccccgg
     3421 ccctctccgc cgctgcaggt ccgcgcacca ggaagctgaa gaagaagaag aacgagaagg
     3481 aggacaagcg gccgcggacc gcgttcacgg ccgagcagct gcagagactc aaggcggagt
     3541 tccaggcaaa ccgctacatc acggagcagc ggcggcagac cctggcccag gaactcagcc
     3601 tcaacgagtc ccagatcaag atctggttcc agaacaagcg cgccaagatc aagaaagcca
     3661 caggcatcaa gaacggcctg gcgctgcacc tcatggccca gggactgtac aaccactcca
     3721 ccaccacggt ccaggacaaa gacgagagcg agggatccgg cgcaacaaac ttctctctgc
     3781 tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggtcagcaaa ggcgaagaag
     3841 acaacatggc atcactgcca gcgacacacg aacttcatat tttcggctcc atcaatggtg
     3901 tggacttcga tatggttgga cagggtactg ggaaccctaa cgatggatat gaggagttga
     3961 acttgaagag taccaagggc gatcttcagt tttcaccttg gattctcgta cctcatatag
     4021 gttatggctt tcatcaatat ctgccctatc ctgacggtat gtcacctttc caagcagcta
     4081 tggttgacgg gagcggttat caggttcacc ggacaatgca gtttgaagat ggagcctctt
     4141 tgaccgtaaa ttatcggtat acttatgagg gcagccatat taaaggcgag gctcaagtta
     4201 aggggactgg gttccccgcg gatggtcccg taatgacaaa cagtctgacg gctgcagact
     4261 ggtgccgctc taagaagaca tatccaaatg acaagacgat tattagcact ttcaagtggt
     4321 catacactac aggaaatggt aaacgctata ggagcactgc ccggaccacg tatactttcg
     4381 cgaagccgat ggcggcaaat tatttgaaaa accaaccgat gtatgtgttc agaaagacag
     4441 aactcaagca ttctaaaact gaactgaact tcaaagagtg gcagaaagcc tttactgatg
     4501 taatgggcat ggacgaactc tataagtaag tcgacctgca gccaagctat cgaattccga
     4561 tcatattcaa taacccttaa tataacttcg tataatgtat gctatacgaa gttatagcgc
     4621 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     4681 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     4741 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     4801 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     4861 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     4921 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     4981 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     5041 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     5101 cttccctgtc ctttaggtct gaagaggagt ttacgtccag ccacgcgtag gatctcgacc
     5161 tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     5221 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     5281 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     5341 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     5401 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     5461 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     5521 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     5581 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     5641 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcctgttgac aattaatcat
     5701 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atgggatcgg
     5761 ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     5821 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     5881 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5941 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     6001 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     6061 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     6121 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     6181 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     6241 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     6301 gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     6361 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     6421 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     6481 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     6541 acgagttctt ctgaggggat caattctcta gagctcgctg atcagcctcg actgtgcctt
     6601 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6661 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     6721 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     6781 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     6841 ggggctcgac tagagcttgc ggaaccctta atataacttc gtataatgta tgctatacga
     6901 agttattagg tccctcgagg ggatcctagc cgccaagtgc cggggccgcg cccgaatccc
     6961 ctaacggcac cgccgccgtc gtctaacggc ccctatatgg gggagaaagc atctgctcca
     7021 aggagggagg gagcgcaggg aaaagagcga gagagacaga aagagagcct cagaatggac
     7081 aatgacgttg aaacgcagca tttttgaaaa gggagaaaga ctcggacagg tgctatcgaa
     7141 aaataagatc cattctctat tcccagtata agggacgaaa ctgcgaactc cttaaagctc
     7201 tatctagcca aaccgcttac gaccttgtat atatttaatt tcaggtaagg aaaacacata
     7261 cgtgtagcga tctctatttg ctggacattt ttattaatct cctttattat tattgttata
     7321 attattttat cccctccccc accgcctcgc tgcccccagc ccagtttcgt tttcgttgcc
     7381 tttttcattt gaatgtcatt gcttctccgg tgcctcccga cccgcatcgc cggccctggt
     7441 ttctctggga cttttctttg tgtgcgagag tgtgtttcct ttcgtgtctg cccacctctt
     7501 ctcccccacc tcccgggtcc cttctgtcgg tctgtctgtt ctgcccccct ttcgttttcc
     7561 ggagacttgt tgagaaatac gaccccacag actgcgagac tgaaccgccg ctacaagcca
     7621 aagattttat tatgttcaga aacctgtagt ctgaaataaa gtgtacactg tgctcacgag
     7681 cgctggcggc cctcctgttt gcgggctcgg gaaggacgtg ggtccgggca cacagcgccc
     7741 cagcgcccct gcgcacgccg ggcggggagc cggccgggcc ttgcgatcac cgcacaacga
     7801 atgcagggcg aaggggaagc gcgacccaga tttgggttta gcctgggtct gaagggtctg
     7861 gcgggacacg cctggtctcc gattgcgttc aagggaactc aaaggccgac aaccacagtc
     7921 ctggcttcgg aaacgctgcc tgggctgcgg ggcagctccc cgcaccggtc caggcccagc
     7981 ccagtggcgg cctcctcctg gcggccgcca ccgcggtgga gctccaattc gccctatagt
     8041 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     8101 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     8161 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg
     8221 taagcg
//
