LOCUS       sequence-365597-        5547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PCREm4-driven Nluc and PmPGK1-driven Fluc 
            expression (PCREm4-Nluc-pA::A2-pA::PmPGK1-Fluc-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: pVH613
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        1107..1138
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             1190..1702
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1763..1780)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1769..1993
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2139..2155)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2139..2155)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2195..2694
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2230..2253)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2627..2646
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2738..4390
                     /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(2804..2822)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     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 tagcctgacg tccgagagcc gtagcctgac gtccgagggt accagcctga cgtccgagag
     1081 ccgtagcctg acgtccgagt actccgtaga gggtatataa tggaagctcg acttccagct
     1141 tggcgagctc ttcgaagcgg aattcaccat gactagtggt ggttctggta tggtcttcac
     1201 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct
     1261 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca
     1321 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta
     1381 tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc
     1441 tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt
     1501 tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg
     1561 caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct
     1621 gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg
     1681 gctgtgcgaa cgcattctgg cggctagcgg atccaccggt gtctagaaag ctttgaggcc
     1741 ggcctgcagg cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1801 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1861 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1921 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1981 gtgggctcta tggtagctta ggaggcggaa agaaccagcg cgggagaccg tcagttggtc
     2041 tccagctata caggtgcacc gctttataat aaaactgata cagagaacgt tatcatgtgt
     2101 gttggttttt tttgtgtgtg cggtttaagg gagcctcggt catagctgtt tcctgtgtaa
     2161 aacgacggcc agcgcgtggt accctcgagc taccgggtag gggaggcgct tttcccaagg
     2221 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     2281 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     2341 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     2401 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca gtagtctcgt gcagatggac
     2461 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     2521 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     2581 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     2641 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctcc
     2701 agcccgagct cggtataggt tcgaagcgga attcaccatg gaagacgcca aaaacataaa
     2761 gaaaggcccg gcgccattct atccgctgga agatggaacc gctggagagc aactgcataa
     2821 ggctatgaag agatacgccc tggttcctgg aacaattgct tttacagatg cacatatcga
     2881 ggtggacatc acttacgctg agtacttcga aatgtccgtt cggttggcag aagctatgaa
     2941 acgatatggg ctgaatacaa atcacagaat cgtcgtatgc agtgaaaact ctcttcaatt
     3001 ctttatgccg gtgttgggcg cgttatttat cggagttgca gttgcgcccg cgaacgacat
     3061 ttataatgaa cgtgaattgc tcaacagtat gggcatttcg cagcctaccg tggtgttcgt
     3121 ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa aagctcccaa tcatccaaaa
     3181 aattattatc atggattcta aaacggatta ccagggattt cagtcgatgt acacgttcgt
     3241 cacatctcat ctacctcccg gttttaatga atacgatttt gtgccagagt ccttcgatag
     3301 ggacaagaca attgcactga tcatgaactc ctctggatct actggtctgc ctaaaggtgt
     3361 cgctctgcct catagaactg cctgcgtgag attctcgcat gccagagatc ctatttttgg
     3421 caatcaaatc attccggata ctgcgatttt aagtgttgtt ccattccatc acggttttgg
     3481 aatgtttact acactcggat atttgatatg tggatttcga gtcgtcttaa tgtatagatt
     3541 tgaagaagag ctgtttctga ggagccttca ggattacaag attcaaagtg cgctgctggt
     3601 gccaacccta ttctccttct tcgccaaaag cactctgatt gacaaatacg atttatctaa
     3661 tttacacgaa attgcttctg gtggcgctcc cctctctaag gaagtcgggg aagcggttgc
     3721 caagaggttc catctgccag gtatcaggca aggatatggg ctcactgaga ctacatcagc
     3781 tattctgatt acacccgagg gggatgataa accgggcgcg gtcggtaaag ttgttccatt
     3841 ttttgaagcg aaggttgtgg atctggatac cgggaaaacg ctgggcgtta atcaaagagg
     3901 cgaactgtgt gtgagaggtc ctatgattat gtccggttat gtaaacaatc cggaagcgac
     3961 caacgccttg attgacaagg atggatggct acattctgga gacatagctt actgggacga
     4021 agacgaacac ttcttcatcg ttgaccgcct gaagtctctg attaagtaca aaggctatca
     4081 ggtggctccc gctgaattgg aatccatctt gctccaacac cccaacatct tcgacgcagg
     4141 tgtcgcaggt cttcccgacg atgacgccgg tgaacttccc gccgccgttg ttgttttgga
     4201 gcacggaaag acgatgacgg aaaaagagat cgtggattac gtcgccagtc aagtaacaac
     4261 cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa gtaccgaaag gtcttaccgg
     4321 aaaactcgac gcaagaaaaa tcagagagat cctcataaag gccaagaagg gcggaaagat
     4381 cgccgtgtaa 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
//
