LOCUS       sequence-366613-        5598 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Tier-2 vector encoding PCREm4-driven Nluc and PmPGK1-driven Fluc 
            expression (A1-pA::PCREm4-Nluc-pA::PmPGK1-Fluc-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 Apr 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: pVH615
FEATURES             Location/Qualifiers
     source          1..5598
                     /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
     primer_bind     complement(1127..1144)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1133..1357
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1473..1504
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             1556..2068
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(2190..2206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2246..2745
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2281..2304)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2678..2697
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2789..4441
                     /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(2855..2873)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    4581..4602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4719..4736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4890..5478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4970..4989)
                     /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 gaggagctct tcgaagcgga attcaccatg actagtgcta gcggatccac
     1081 cggtgtctag aaagctttga ggccggcctg caggcccgct gatcagcctc gactgtgcct
     1141 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     1201 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     1261 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     1321 aatagcaggc atgctgggga tgcggtgggc tctatggtag cttaggaggc ggaaagaacc
     1381 agcgcgtagc ctgacgtccg agagccgtag cctgacgtcc gagggtacca gcctgacgtc
     1441 cgagagccgt agcctgacgt ccgagtactc cgtagagggt atataatgga agctcgactt
     1501 ccagcttggc gagctcttcg aagcggaatt caccatgact agtggtggtt ctggtatggt
     1561 cttcacactc gaagatttcg ttggggactg gcgacagaca gccggctaca acctggacca
     1621 agtccttgaa cagggaggtg tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc
     1681 gatccaaagg attgtcctga gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat
     1741 cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt
     1801 gtaccctgtg gatgatcatc actttaaggt gatcctgcac tatggcacac tggtaatcga
     1861 cggggttacg ccgaacatga tcgactattt cggacggccg tatgaaggca tcgccgtgtt
     1921 cgacggcaaa aagatcactg taacagggac cctgtggaac ggcaacaaaa ttatcgacga
     1981 gcgcctgatc aaccccgacg gctccctgct gttccgagta accatcaacg gagtgaccgg
     2041 ctggcggctg tgcgaacgca ttctggcggc tagcggatcc accggtgtct agaaagctat
     2101 acaggtgcac cgctttataa taaaactgat acagagaacg ttatcatgtg tgttggtttt
     2161 ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt ttcctgtgta aaacgacggc
     2221 cagcgcgtgg taccctcgag ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     2281 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     2341 acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg
     2401 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     2461 aggacgtgac aaatggaagt agcacgtctc agtagtctcg tgcagatgga cagcaccgct
     2521 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     2581 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     2641 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     2701 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctc cagcccgagc
     2761 tcggtatagg ttcgaagcgg aattcaccat ggaagacgcc aaaaacataa agaaaggccc
     2821 ggcgccattc tatccgctgg aagatggaac cgctggagag caactgcata aggctatgaa
     2881 gagatacgcc ctggttcctg gaacaattgc ttttacagat gcacatatcg aggtggacat
     2941 cacttacgct gagtacttcg aaatgtccgt tcggttggca gaagctatga aacgatatgg
     3001 gctgaataca aatcacagaa tcgtcgtatg cagtgaaaac tctcttcaat tctttatgcc
     3061 ggtgttgggc gcgttattta tcggagttgc agttgcgccc gcgaacgaca tttataatga
     3121 acgtgaattg ctcaacagta tgggcatttc gcagcctacc gtggtgttcg tttccaaaaa
     3181 ggggttgcaa aaaattttga acgtgcaaaa aaagctccca atcatccaaa aaattattat
     3241 catggattct aaaacggatt accagggatt tcagtcgatg tacacgttcg tcacatctca
     3301 tctacctccc ggttttaatg aatacgattt tgtgccagag tccttcgata gggacaagac
     3361 aattgcactg atcatgaact cctctggatc tactggtctg cctaaaggtg tcgctctgcc
     3421 tcatagaact gcctgcgtga gattctcgca tgccagagat cctatttttg gcaatcaaat
     3481 cattccggat actgcgattt taagtgttgt tccattccat cacggttttg gaatgtttac
     3541 tacactcgga tatttgatat gtggatttcg agtcgtctta atgtatagat ttgaagaaga
     3601 gctgtttctg aggagccttc aggattacaa gattcaaagt gcgctgctgg tgccaaccct
     3661 attctccttc ttcgccaaaa gcactctgat tgacaaatac gatttatcta atttacacga
     3721 aattgcttct ggtggcgctc ccctctctaa ggaagtcggg gaagcggttg ccaagaggtt
     3781 ccatctgcca ggtatcaggc aaggatatgg gctcactgag actacatcag ctattctgat
     3841 tacacccgag ggggatgata aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc
     3901 gaaggttgtg gatctggata ccgggaaaac gctgggcgtt aatcaaagag gcgaactgtg
     3961 tgtgagaggt cctatgatta tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt
     4021 gattgacaag gatggatggc tacattctgg agacatagct tactgggacg aagacgaaca
     4081 cttcttcatc gttgaccgcc tgaagtctct gattaagtac aaaggctatc aggtggctcc
     4141 cgctgaattg gaatccatct tgctccaaca ccccaacatc ttcgacgcag gtgtcgcagg
     4201 tcttcccgac gatgacgccg gtgaacttcc cgccgccgtt gttgttttgg agcacggaaa
     4261 gacgatgacg gaaaaagaga tcgtggatta cgtcgccagt caagtaacaa ccgcgaaaaa
     4321 gttgcgcgga ggagttgtgt ttgtggacga agtaccgaaa ggtcttaccg gaaaactcga
     4381 cgcaagaaaa atcagagaga tcctcataaa ggccaagaag ggcggaaaga tcgccgtgta
     4441 agctagcgga tccaccggtg tctagaaagc tcagaaatta tgagagcgtt taataaagta
     4501 ttgactatgg ttgacaaaac gtgtgttgga gttttttgtg tgctttgtca tcaactgagc
     4561 cagtaggtcg agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4621 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4681 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggattat caaaaaggat
     5521 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5581 gtaaacttgg tctgacag
//
