LOCUS       sequence-364521-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven mTagBFP2 expression 
            (PmPGK1-mTagBFP2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  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 5778)
  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: pVH231-Tier1-PmPGK1-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1982..2481
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2017..2040)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2414..2433
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2546..3253
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     primer_bind     complement(3308..3325)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3314..3538
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3584..4012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3671..3690)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3881..3902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4021..4041)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4026..4355
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4206..4341
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4268..4287
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4422..5216
                     /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(4476..4495)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5086..5105
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5390..5511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5427..5446)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5481..5500
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5560..5576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5560..5576)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5573..5595)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5584..5600
                     /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(5608..5614,5615..5632,5633..5638))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5653..5674
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgacgtcg acggatcggg agatctccac gcgtggtacc ctcgagctac
     1981 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
     2041 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
     2101 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     2161 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     2221 cgtctcagta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     2281 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     2341 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     2401 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     2461 tcctcatctc cgggcctttc gacctccagc ccgagctcgg tataggttcg aagcggaatt
     2521 caccatgact agtggtggtt ctggtgtgtc taagggcgaa gagctgatta aggagaacat
     2581 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     2641 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     2701 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     2761 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     2821 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     2881 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     2941 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     3001 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     3061 tctgatcgca aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     3121 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     3181 ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg
     3241 gcacaaactt aatgctagcg gatccaccgg tgtctagaaa gctttgaggc cggcctgcag
     3301 ggatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3361 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3421 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3481 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3541 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3601 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3661 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3721 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3781 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3841 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3901 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3961 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     4021 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4081 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4141 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4201 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4261 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4321 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4381 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4441 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4501 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4561 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4621 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4681 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4741 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4801 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4861 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4921 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4981 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     5041 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     5101 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     5161 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     5221 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5281 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5341 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     5401 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5461 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5521 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5581 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5641 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5701 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5761 cggtttgcgt attgggcg
//
