LOCUS       sequence-419012-        7250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector to tag endogenous pig HES7 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7250)
  AUTHORS   Conrad JV, Meyer S, Ramesh PS, Neira JA, Rusteika M, Mamott D, 
            Duffin B, Bautista M, Zhang J, Hiles E, Higgins EM, Steill J, 
            Freeman J, Ni Z, Liu S, Ungrin M, Rancourt D, Clegg DO, Stewart R, 
            Thomson JA, Chu LF
  TITLE     Efficient derivation of transgene-free porcine induced pluripotent 
            stem cells enables in vitro modeling of species-specific 
            developmental timing.
  JOURNAL   Stem Cell Reports. 2023 Dec 12;18(12):2328-2343. doi: 
            10.1016/j.stemcr.2023.10.009. Epub 2023 Nov 9.
  PUBMED    37949072
REFERENCE   2  (bases 1 to 7250)
  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"; doi: 
            "10.1016/j.stemcr.2023.10.009"; journalName: "Stem Cell Reports"; 
            date: "2023-12-12- 12"; volume: "18"; issue: "12"; pages: 
            "2328-2343"
COMMENT     Sequence Label: pHES7-NLuc-2A-tdTomato
FEATURES             Location/Qualifiers
     source          1..7250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             839..1351
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1361..1417
                     /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"
     regulatory      1436..1445
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1442..2872
                     /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    2994..3115
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3031..3050)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3085..3104
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3128..3161)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        3180..3679
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3215..3238)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3612..3631
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3696..3743
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3762..4565
                     /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(3825..3844)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4435..4454
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(4704..4737)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     rep_origin      complement(5495..6083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5575..5594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6254..7045,7046..7114))
                     /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"
     promoter        complement(7115..7219)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatga tactggagtt cgccgtgggc tacttgaggg agcgaagccg ggtggagccc
       61 cccggtacag cgcgggggtg gggcagcaga gggagggagg gaggggggat tcacccgggc
      121 ccgggccggg gtaggagggc cggtggacgg tgggctcggg gagtggcagg ctctactacg
      181 aggcgaggtt aataataaca cccgcgcgtg tctgtctctg tgtctccctc attttctccc
      241 tctgtcggtc catctggctt cctgtctctg tgcgcctgtc cggcctcggt atctctgcgc
      301 cccgcccctt ccctcacccc tgctgggacc cctgccctgc ccgcccsccc tcccgccggt
      361 cgcaaccgct gccgcggctc caggggttcc ccggtccccc gcccaggacg ccgaggcgct
      421 cgccagctgc tacttgtccg gcttccgcga gtgcctgctt cgcctggcgg ccttcgcgca
      481 cgacgccagc ccggccgccc gcgcccagct cttctccgcg ctgcacggat acctgcgccc
      541 caagccgccc cggcccgaac cggtagatcc aaggccccag gcgccgcgcc caccgctgga
      601 ccccgccgcc ccagcgcctg gcccggcgct gcaccagcgc ccgccagtgc acaagggccc
      661 ccctagcccg cgctgcgcat ggtccccggc cccctgctcc ccccgcgccg gggattccgg
      721 cgcgccggcc cccctcaccg gactgctgcc gccgccgccg ccgcctcaca gacaagaggg
      781 ggcgcccaag gccccgccgc ccccgccgcc cgctttctgg agaccttggc ccggatccat
      841 ggtcttcaca ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga
      901 ccaagtcctt gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac
      961 tccgatccaa aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat
     1021 catcccgtat gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt
     1081 ggtgtaccct gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat
     1141 cgacggggtt acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt
     1201 gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga
     1261 cgagcgcctg atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac
     1321 cggctggcgg ctgtgcgaac gcattctggc gggaagcgga gctactaact tcagcctgct
     1381 gaagcaggct ggagacgtgg aggagaaccc tggacctacc cggggggtac cggtcgccac
     1441 catggtgagc aagggcgagg aggtcatcaa agagttcatg cgcttcaagg tgcgcatgga
     1501 gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     1561 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
     1621 catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc ccgccgacat
     1681 ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     1741 cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat
     1801 ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa tgcagaagaa
     1861 gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg
     1921 cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg agttcaagac
     1981 catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg acaccaagct
     2041 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gctccgaggg
     2101 ccgccaccac ctgttcctgg ggcatggcac cggcagcacc ggcagcggca gctccggcac
     2161 cgcctcctcc gaggacaaca acatggccgt catcaaagag ttcatgcgct tcaaggtgcg
     2221 catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2281 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc
     2341 ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc
     2401 cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     2461 gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac
     2521 gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca
     2581 gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct
     2641 gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt
     2701 caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac
     2761 caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc
     2821 cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt aggcggccgc
     2881 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2941 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     3001 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3061 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     3121 agatcttgaa gttcctatac tttctagaga ataggaactt cacgaagtta tattctaccg
     3181 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     3241 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     3301 ccaaccggct ccgttctttg gtggccccgt cgcgccacct tctactcctc ccctagtcag
     3361 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     3421 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     3481 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     3541 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     3601 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     3661 ctcatctccg ggcctttcga cctgcagcag cacgtgttga caattaatca tcggcatagt
     3721 atatcggcat agtataatac gacaaggtga ggaactaaac catgggatcg gccattgaac
     3781 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3841 gggcacaaca gacgatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3901 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3961 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4021 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4081 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4141 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4201 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4261 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4321 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4381 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4441 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4501 acggtatcgc cgcccccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4561 tctgagcggg actctggggt tcgaataaag accgaccaag cgacgtctga gagctccctg
     4621 gcgaattcgg taccaataaa agagctttat tttcatgatc tgtgtgttgg tttttgtgtg
     4681 cggcgcgata acttcgtata gctgaagttc ctatactttc tagagaatag gaacttcgct
     4741 agcgctttga ggggtggggt gggggcgggg gctggggggt ggggtagaga ctccagcccg
     4801 agggcagcag agggacccgg gcgtcggggc gaggaggtgt tggggagggc ggcggggcgc
     4861 gcgggcgtag cgcgcgggtg agatgtggtc tatattagag tatctatata aatatatatt
     4921 tccctggttc ctgtcccttc tccctgccct ccttgcgtct aggattgtac cctctgcccc
     4981 cggcccagcc ccagtcccct cccgagtccc tagtgcatgg aataaagtgg ttattcaatc
     5041 cccgtgtgtc ccctagccag gggcctgcct ttatctcggc gtccacgccc actttccttc
     5101 cgcttctgtc tcccaacccc cagtcctgct ctccttggcc caagccgcgg ggcacacagg
     5161 taagtaaaga gcagagcgga ggctgaggtt ggactgaagc cggatggcaa acacttgaga
     5221 tagggtaggg agagaaggga taggagcctc taagaaaaag ccagatatag aaaggaggga
     5281 aaaaaaaaaa aaaatgaata aatgaaaata aaaccgactc tggtgggatt cgaacccaca
     5341 acctttgaat cgctctttcg tcactagaag tccaatgcgc tatccattgc gccacagagc
     5401 cacctggcgg gcggcggcat ccgatcggaa agaacatgtg agcaaaaggc cagcaaaagg
     5461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccac ccccctgacg
     5521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6241 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6301 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6361 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6421 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6481 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6541 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6601 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6661 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6721 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6781 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6841 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6901 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6961 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7021 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7081 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7141 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7201 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
