LOCUS       sequence-364523-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven Fluc (PmPGK1-Fluc-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  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 6702)
  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: pVH608-Tier1-PmPGK1-Fluc
FEATURES             Location/Qualifiers
     source          1..6702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(61..78)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(232..820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(312..331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(991..1782,1783..1851))
                     /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     1614..1633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1852..1956)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2030..2529
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2065..2088)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2462..2481
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2573..4225
                     /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(2639..2657)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(4280..4297)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4286..4510
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4556..4984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4643..4662)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4853..4874
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4993..5013)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4998..5327
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5178..5313
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5240..5259
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5394..6188
                     /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(5448..5467)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6058..6077
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6362..6483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6399..6418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6453..6472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6532..6548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6532..6548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6545..6567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6556..6572
                     /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(6580..6586,6587..6604,6605..6610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6625..6646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
       61 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
      121 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
      181 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
      241 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      301 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      361 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      421 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      481 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      541 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
      601 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
      661 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
      721 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
      781 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
      841 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
      901 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
      961 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     1021 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     1081 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     1141 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1201 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1261 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1321 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     1381 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1441 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1501 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1561 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     1621 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1681 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     1741 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     1801 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     1861 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     1921 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     1981 tgacgtcgac ggatcgggag atctccacgc gtggtaccct cgagctaccg ggtaggggag
     2041 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     2101 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     2161 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     2221 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcagtagt
     2281 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     2341 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     2401 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     2461 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     2521 ggcctttcga cctccagccc gagctcggta taggttcgaa gcggaattca ccatggaaga
     2581 cgccaaaaac ataaagaaag gcccggcgcc attctatccg ctggaagatg gaaccgctgg
     2641 agagcaactg cataaggcta tgaagagata cgccctggtt cctggaacaa ttgcttttac
     2701 agatgcacat atcgaggtgg acatcactta cgctgagtac ttcgaaatgt ccgttcggtt
     2761 ggcagaagct atgaaacgat atgggctgaa tacaaatcac agaatcgtcg tatgcagtga
     2821 aaactctctt caattcttta tgccggtgtt gggcgcgtta tttatcggag ttgcagttgc
     2881 gcccgcgaac gacatttata atgaacgtga attgctcaac agtatgggca tttcgcagcc
     2941 taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct
     3001 cccaatcatc caaaaaatta ttatcatgga ttctaaaacg gattaccagg gatttcagtc
     3061 gatgtacacg ttcgtcacat ctcatctacc tcccggtttt aatgaatacg attttgtgcc
     3121 agagtccttc gatagggaca agacaattgc actgatcatg aactcctctg gatctactgg
     3181 tctgcctaaa ggtgtcgctc tgcctcatag aactgcctgc gtgagattct cgcatgccag
     3241 agatcctatt tttggcaatc aaatcattcc ggatactgcg attttaagtg ttgttccatt
     3301 ccatcacggt tttggaatgt ttactacact cggatatttg atatgtggat ttcgagtcgt
     3361 cttaatgtat agatttgaag aagagctgtt tctgaggagc cttcaggatt acaagattca
     3421 aagtgcgctg ctggtgccaa ccctattctc cttcttcgcc aaaagcactc tgattgacaa
     3481 atacgattta tctaatttac acgaaattgc ttctggtggc gctcccctct ctaaggaagt
     3541 cggggaagcg gttgccaaga ggttccatct gccaggtatc aggcaaggat atgggctcac
     3601 tgagactaca tcagctattc tgattacacc cgagggggat gataaaccgg gcgcggtcgg
     3661 taaagttgtt ccattttttg aagcgaaggt tgtggatctg gataccggga aaacgctggg
     3721 cgttaatcaa agaggcgaac tgtgtgtgag aggtcctatg attatgtccg gttatgtaaa
     3781 caatccggaa gcgaccaacg ccttgattga caaggatgga tggctacatt ctggagacat
     3841 agcttactgg gacgaagacg aacacttctt catcgttgac cgcctgaagt ctctgattaa
     3901 gtacaaaggc tatcaggtgg ctcccgctga attggaatcc atcttgctcc aacaccccaa
     3961 catcttcgac gcaggtgtcg caggtcttcc cgacgatgac gccggtgaac ttcccgccgc
     4021 cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg attacgtcgc
     4081 cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg acgaagtacc
     4141 gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca taaaggccaa
     4201 gaagggcgga aagatcgccg tgtaagctag cggatccacc ggtgtctaga aagctttgag
     4261 gccggcctgc agggatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4321 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4381 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4441 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4501 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     4561 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4621 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4681 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4741 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4801 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4861 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4921 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4981 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     5041 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     5101 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     5161 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     5221 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     5281 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     5341 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     5401 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     5461 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     5521 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     5581 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     5641 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     5701 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     5761 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     5821 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     5881 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     5941 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     6001 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     6061 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     6121 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     6181 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     6241 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     6301 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     6361 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6421 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6481 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     6541 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6601 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6661 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg ca
//
