LOCUS       sequence-366619-        5547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PmPGK1-driven Fluc and PCREm4-driven Nluc 
            expression (PmPGK1-Fluc-pA::A2-pA::PCREm4-Nluc-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5547)
  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 5547)
  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: pVH616
FEATURES             Location/Qualifiers
     source          1..5547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1039..1538
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1074..1097)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1471..1490
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1582..3234
                     /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(1648..1666)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(3295..3312)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3301..3525
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3671..3687)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3671..3687)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3795..3826
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             3878..4390
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     protein_bind    4530..4551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4668..4685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4839..5427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4919..4938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc ggctcccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccaaga tctccacgcg
     1021 tggtaccctc gagctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg
     1081 ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt
     1141 ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     1201 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     1261 gacaaatgga agtagcacgt ctcagtagtc tcgtgcagat ggacagcacc gctgagcaat
     1321 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     1381 ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg
     1441 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     1501 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctccagcccg agctcggtat
     1561 aggttcgaag cggaattcac catggaagac gccaaaaaca taaagaaagg cccggcgcca
     1621 ttctatccgc tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac
     1681 gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac
     1741 gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat
     1801 acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg
     1861 ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa
     1921 ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg
     1981 caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat
     2041 tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct
     2101 cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca
     2161 ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga
     2221 actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg
     2281 gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc
     2341 ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt
     2401 ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc
     2461 ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct
     2521 tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg
     2581 ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc
     2641 gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt
     2701 gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga
     2761 ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac
     2821 aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc
     2881 atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa
     2941 ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc
     3001 gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg
     3061 acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc
     3121 ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga
     3181 aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaagctagc
     3241 ggatccaccg gtgtctagaa agctttgagg ccggcctgca ggcccgctga tcagcctcga
     3301 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3361 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3421 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3481 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggtagct taggaggcgg
     3541 aaagaaccag cgcgggagac cgtcagttgg tctccagcta tacaggtgca ccgctttata
     3601 ataaaactga tacagagaac gttatcatgt gtgttggttt tttttgtgtg tgcggtttaa
     3661 gggagcctcg gtcatagctg tttcctgtgt aaaacgacgg ccagcgcgta gcctgacgtc
     3721 cgagagccgt agcctgacgt ccgagggtac cagcctgacg tccgagagcc gtagcctgac
     3781 gtccgagtac tccgtagagg gtatataatg gaagctcgac ttccagcttg gcgagctctt
     3841 cgaagcggaa ttcaccatga ctagtggtgg ttctggtatg gtcttcacac tcgaagattt
     3901 cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg
     3961 tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct
     4021 gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag
     4081 cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca
     4141 tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat
     4201 gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac
     4261 tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga
     4321 cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg
     4381 cattctggcg gctagcggat ccaccggtgt ctagaaagct cagaaattat gagagcgttt
     4441 aataaagtat tgactatggt tgacaaaacg tgtgttggag ttttttgtgt gctttgtcat
     4501 caactgagcc agtaggtcga gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4561 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4621 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4681 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4741 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4801 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4861 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4921 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4981 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5041 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5101 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5161 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5221 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5281 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5341 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5401 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggattatc
     5461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5521 tatatatgag taaacttggt ctgacag
//
