LOCUS       sequence-396542-        6725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate compatible plasmid with mCherry and Neo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6725)
  AUTHORS   Maimaitili M, Chen M, Febbraro F, Ucuncu E, Kelly R, Niclis JC, 
            Christiansen JR, Mermet-Joret N, Niculescu D, Lauritsen J, Iannielli
            A, Klaestrup IH, Jensen UB, Qvist P, Nabavi S, Broccoli V, Nykjaer 
            A, Romero-Ramos M, Denham M
  TITLE     Enhanced production of mesencephalic dopaminergic neurons from 
            lineage-restricted human undifferentiated stem cells.
  JOURNAL   Nat Commun. 2023 Dec 5;14(1):7871. doi: 10.1038/s41467-023-43471-0.
  PUBMED    38052784
REFERENCE   2  (bases 1 to 6725)
  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: "Nat 
            Commun."; date: "2023-12-5"; volume: "14(1):7871. doi"; pages: " 
            10.1038/s41467-023-43471-0"
COMMENT     Sequence Label: pBS-BsmbI-LoxP-Ef1a-EM7-mCherry-Neo-PolyA-LoxP
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(471..989)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY
                     RGRHRGGAPIRPIVSRITRAHWPSFYNVVTGKTLALPNLIALQHIPLSPAGVIAKRPAP
                     IALPNSCAA"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     854..870
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        880..898
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(946..964)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(985..1001)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1009..1025
                     /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(1033..1039,1040..1057,1058..1063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1078..1099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     1156..1172
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1156..1172
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(1192..1225)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1312..2490
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1543..2481
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2438..2458
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2516..2563
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2588..2608
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2609..3316
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2754..2772)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3022..3041
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3335..3388
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3395..4198
                     /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
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3458..3477)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4068..4087
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4212..4436
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4437..4470)
                     /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(4491..4507)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4492..4508)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4537..4555)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4576..4592)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4600..4616
                     /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(4624..4630,4631..4648,4649..4654))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4669..4690
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4807..4824)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4978..5566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5058..5077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5737..6528,6529..6597))
                     /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     6360..6379
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6598..6702)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgcgata agagacggtg gtggcgccgc tacagggcgc gtcccattcg
      721 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
      781 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
      841 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
      901 gaattgggta ccgggccccc cctcgaggtc ctccagcttt tgttcccttt agtgagggtt
      961 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1021 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1081 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccact gcatgacgtc
     1141 tccacaagcg gccgctctag aactagtgga tcccccgggc tgcaggaatt cataacttcg
     1201 tatagcatac attatacgaa gttataagct ttgcaaagat ggataaagtt ttaaacagag
     1261 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
     1321 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     1381 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     1441 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     1501 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     1561 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     1621 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     1681 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     1741 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     1801 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     1861 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     1921 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     1981 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     2041 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     2101 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     2161 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     2221 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     2281 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     2341 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     2401 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     2461 agtttttttc ttccatttca ggtgtcgtga ggaatttcga catttaaatc ccggggttga
     2521 caattaatca tcggcatagt atatcggcat agtataatac gactcactat acgccaccat
     2581 gactgctcca aagaagaagc gtaaggtaat ggtgagcaag ggcgaggagg ataacatggc
     2641 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     2701 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     2761 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     2821 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     2881 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     2941 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     3001 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     3061 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     3121 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     3181 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     3241 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     3301 ggacgagctg tacaagggcg gcgggtccgg aggagagggc agaggaagtc ttctaacatg
     3361 cggtgacgtg gaggagaatc ctggcccagt cgagatggga tcggccattg aacaagatgg
     3421 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3481 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3541 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3601 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3661 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3721 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3781 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3841 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3901 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     3961 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4021 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     4081 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4141 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg
     4201 agatccatat gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4261 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4321 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4381 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggataa
     4441 cttcgtatag catacattat acgaagttat gaattcgata tcaagcttat cgataccgtc
     4501 gacctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     4561 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4621 acacaacata cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4681 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4801 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4861 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5401 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5641 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5701 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5761 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5821 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5881 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5941 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6001 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6061 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6121 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6181 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6241 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6301 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6361 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6421 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6481 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6541 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6601 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6661 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6721 gccac
//
