LOCUS       sequence-365003-        6067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven TtgA expression 
            (PmPGK1-TtgA-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 6067)
  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: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pVH237-2-Tier1-PmPGK1-TtgA
FEATURES             Location/Qualifiers
     source          1..6067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..24)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(178..766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(258..277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(937..1728,1729..1797))
                     /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     1560..1579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1798..1902)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1976..2475
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2011..2034)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2408..2427
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3303..3536
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(3591..3608)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3597..3821
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3867..4295
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3954..3973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4164..4185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4304..4324)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4309..4638
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4489..4624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4551..4570
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4705..5499
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4759..4778)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5369..5388
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5673..5794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5710..5729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5764..5783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5843..5859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5843..5859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5856..5878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5867..5883
                     /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(5891..5897,5898..5915,5916..5921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5936..5957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
       61 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      121 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      181 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      241 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      301 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      361 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      421 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      481 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      541 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      601 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      661 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      721 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
      781 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
      841 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
      901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
      961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1081 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     1381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     1441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     1501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     1561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     1621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     1681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     1741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     1801 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     1861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     1921 gccacctgac gtcgacggat cgggagatct ccacgcgtgg taccctcgag ctaccgggta
     1981 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     2041 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     2101 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     2161 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     2221 agtagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     2281 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     2341 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     2401 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     2461 tctccgggcc tttcgacctc cagcccgagc tcggtatagg ttcgaagcgg aattccacca
     2521 tggtccgtcg aaccaaagaa gaagcccagg aaacccgtgc ccagatcatc gaggcggcgg
     2581 aaagggcctt ctacaagcgc ggggttgcgc gaaccaccct ggccgacatc gccgaactgg
     2641 cgggtgtgac gcggggggcg atctactggc acttcaacaa caaggccgag ctggtgcagg
     2701 cgttgctcga cagcctgcac gagacccatg accatttggc gcgggcgagc gaaagcgagg
     2761 atgaagtcga cccgcttggc tgcatgcgca agctgctgtt gcaagtgttc aacgagctgg
     2821 tgctcgatgc ccgaacccgt cgtatcaatg aaatcctgca tcacaagtgc gagttcaccg
     2881 atgacatgtg tgaaattcgc cagcagcgcc agagtgcggt gctggattgc cacaagggca
     2941 tcaccctggc gctggccaat gccgtacgcc ggggccagtt gcctggcgag ctggatgccg
     3001 agcgagcggc ggtggccatg tttgcctatg tcgatggcct gatcaggcgc tggctattgc
     3061 tgcctgacag tgtcgatctg ttgggtgatg tcgaaaaatg ggtcgacacc gggctggata
     3121 tgctgcgctt gagcccggct ctgcgcaaat ccgcgtacag ccgcgcgcgt acgaaaaaca
     3181 attacgggtc taccatcgag ggcctgctcg atctcccgga cgacgacgcc cccgaagagg
     3241 cggggctggc ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc agactgtcga
     3301 cggccccccc gaccgatgtc agcctggggg acgagctcca cttagacggc gaggacgtgg
     3361 cgatggcgca tgccgacgcg ctagacgatt tcgatctgga catgttgggg gacggggatt
     3421 ccccgggtcc gggatttacc ccccacgact ccgcccccta cggcgctctg gatatggccg
     3481 acttcgagtt tgagcagatg tttaccgatg cccttggaat tgacgagtac ggtggggcta
     3541 gcggatccac cggtgtctag aaagctttga ggccggcctg cagggatcag cctcgactgt
     3601 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3661 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3721 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3781 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3841 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3901 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3961 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4021 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4081 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4141 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4201 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4261 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4321 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4381 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4441 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4501 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4561 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4621 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     4681 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4741 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4801 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4861 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4921 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4981 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     5041 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     5101 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     5161 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     5221 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5281 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5341 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5401 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5461 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5521 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5581 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5641 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5701 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5761 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5821 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5881 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5941 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6001 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6061 gctcttc
//
