LOCUS       sequence-364605-        5772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven eYFP expression 
            (PmPGK1-eYFP-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  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 5772)
  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: pVH233-Tier1-PmPGK1-eYFP
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(135..723)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(215..234)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(894..1685,1686..1754))
                     /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     1517..1536
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1755..1859)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1933..2432
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1968..1991)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2365..2384
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2485..2487,2488..3198)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2527..2548)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2788..2807)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3135..3156
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3253..3270)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3259..3483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3529..3957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3616..3635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3826..3847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3966..3986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3971..4300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4151..4286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4213..4232
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4367..5161
                     /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(4421..4440)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5031..5050
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5335..5456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5372..5391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5426..5445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5505..5521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5505..5521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5518..5540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5529..5545
                     /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(5553..5559,5560..5577,5578..5583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5598..5619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5736..5753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
       61 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      121 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      181 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      241 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      301 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      361 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      421 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      481 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      541 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      601 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      661 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      721 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      781 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      841 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      901 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      961 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1021 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1081 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1141 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1201 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1261 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1321 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1381 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1441 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1501 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1561 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1621 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1681 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1741 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1801 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1861 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctcca cgcgtggtac
     1921 cctcgagcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag
     1981 cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat
     2041 ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc
     2101 ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa
     2161 tggaagtagc acgtctcagt agtctcgtgc agatggacag caccgctgag caatggaagc
     2221 gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga
     2281 ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg
     2341 cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc
     2401 tgttctcctc ttcctcatct ccgggccttt cgacctccag cccgagctcg gtataggttc
     2461 gaagcggaat tcaccatgac tagtgtgagc aagggcgagg agctgttcac cggggtggtg
     2521 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2581 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2641 ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc
     2701 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2761 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2821 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2881 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2941 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     3001 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3061 gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac
     3121 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3181 atggacgagc tgtacaaggc tagcggatcc accggtgtct agaaagcttt gaggccggcc
     3241 tgcagggatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3301 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3361 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3421 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3481 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3541 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3601 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3661 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3721 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3781 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3841 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3901 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3961 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4021 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4081 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4141 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4201 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     4261 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     4321 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4381 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4441 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4501 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4561 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4621 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4681 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4741 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4801 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4861 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4921 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4981 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     5041 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     5101 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5161 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     5221 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5281 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     5341 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5401 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5461 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5521 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5581 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5641 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5701 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5761 gtcgttcggc tg
//
