LOCUS       sequence-365600-        5598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PmPGK1-driven Fluc and PCREm4-driven Nluc 
            expression (A1-pA::PmPGK1-Fluc-pA::PCREm4-Nluc-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5598)
  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 5598)
  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: pVH617
FEATURES             Location/Qualifiers
     source          1..5598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     618..637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(856..960)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1121..1138)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1127..1351
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1399..1898
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1434..1457)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1831..1850
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1942..3594
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(2008..2026)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(3716..3732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3716..3732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3840..3871
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             3923..4435
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     protein_bind    4575..4596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4713..4730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4884..5472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4964..4983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5593..5598,1..786,787..855))
                     /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"
ORIGIN
        1 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
       61 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
      121 tgcaatgata ccgcggctcc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
      181 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
      241 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
      301 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      361 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      421 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      481 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      541 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      601 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      661 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      721 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      781 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      841 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      901 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      961 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc aagatctcca cgcgtggtac
     1021 cctcgaggag ctcttcgaag cggaattcac catgactagt gctagcggat ccaccggtgt
     1081 ctagaaagct ttgaggccgg cctgcaggcc cgctgatcag cctcgactgt gccttctagt
     1141 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1201 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1261 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1321 aggcatgctg gggatgcggt gggctctatg gtagcttagg aggcggaaag aaccagcgcg
     1381 tggtaccctc gagctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg
     1441 ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt
     1501 ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     1561 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     1621 gacaaatgga agtagcacgt ctcagtagtc tcgtgcagat ggacagcacc gctgagcaat
     1681 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     1741 ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg
     1801 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     1861 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctccagcccg agctcggtat
     1921 aggttcgaag cggaattcac catggaagac gccaaaaaca taaagaaagg cccggcgcca
     1981 ttctatccgc tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac
     2041 gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac
     2101 gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat
     2161 acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg
     2221 ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa
     2281 ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg
     2341 caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat
     2401 tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct
     2461 cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca
     2521 ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga
     2581 actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg
     2641 gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc
     2701 ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt
     2761 ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc
     2821 ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct
     2881 tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg
     2941 ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc
     3001 gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt
     3061 gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga
     3121 ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac
     3181 aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc
     3241 atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa
     3301 ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc
     3361 gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg
     3421 acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc
     3481 ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga
     3541 aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaagctagc
     3601 ggatccaccg gtgtctagaa agctatacag gtgcaccgct ttataataaa actgatacag
     3661 agaacgttat catgtgtgtt ggtttttttt gtgtgtgcgg tttaagggag cctcggtcat
     3721 agctgtttcc tgtgtaaaac gacggccagc gcgtagcctg acgtccgaga gccgtagcct
     3781 gacgtccgag ggtaccagcc tgacgtccga gagccgtagc ctgacgtccg agtactccgt
     3841 agagggtata taatggaagc tcgacttcca gcttggcgag ctcttcgaag cggaattcac
     3901 catgactagt ggtggttctg gtatggtctt cacactcgaa gatttcgttg gggactggcg
     3961 acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt
     4021 tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg
     4081 gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg
     4141 ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat
     4201 cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg
     4261 acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct
     4321 gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt
     4381 ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcggctag
     4441 cggatccacc ggtgtctaga aagctcagaa attatgagag cgtttaataa agtattgact
     4501 atggttgaca aaacgtgtgt tggagttttt tgtgtgcttt gtcatcaact gagccagtag
     4561 gtcgagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4621 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4681 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4741 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4801 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4861 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4921 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4981 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5041 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5101 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5161 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5221 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5281 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5341 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5401 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5461 aaaggatctc aagaagatcc tttgatcttt tctacgggga ttatcaaaaa ggatcttcac
     5521 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5581 ttggtctgac agttacca
//
