LOCUS       sequence-364522-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PmPGK1-driven mRuby2 expression 
            (PmPGK1-mRuby2-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: pVH232-Tier1-PmPGK1-mRuby2
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..35)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(189..777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(269..288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(948..1739,1740..1808))
                     /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     1571..1590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1809..1913)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1987..2486
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2022..2045)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2419..2438
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2551..3258
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     primer_bind     complement(3313..3330)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3319..3543
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3589..4017
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3676..3695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3886..3907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4026..4046)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4031..4360
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4211..4346
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4273..4292
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4427..5221
                     /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(4481..4500)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5091..5110
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5395..5516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5432..5451)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5486..5505
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5565..5581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5565..5581)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5578..5600)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5589..5605
                     /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(5613..5619,5620..5637,5638..5643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5658..5679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
       61 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      121 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      601 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
      661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
      781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
      841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
      901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
      961 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1021 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1081 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1141 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1201 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     1261 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     1321 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     1381 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     1441 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     1501 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     1561 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     1621 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     1681 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     1741 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     1801 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     1861 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1921 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tccacgcgtg gtaccctcga
     1981 gctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     2041 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     2101 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     2161 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     2221 tagcacgtct cagtagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     2281 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     2341 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     2401 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     2461 cctcttcctc atctccgggc ctttcgacct ccagcccgag ctcggtatag gttcgaagcg
     2521 gaattcacca tgactagtgg tggttctggt gtgtctaagg gcgaagagct gatcaaggaa
     2581 aatatgcgta tgaaggtggt catggaaggt tcggtcaacg gccaccaatt caaatgcaca
     2641 ggtgaaggag aaggcaatcc gtacatggga actcaaacca tgaggatcaa agtcatcgag
     2701 ggaggacccc tgccatttgc ctttgacatt cttgccacgt cgttcatgta tggcagccgt
     2761 acttttatca agtacccgaa aggcattcct gatttcttta aacagtcctt tcctgagggt
     2821 tttacttggg aaagagttac gagatacgaa gatggtggag tcgtcaccgt catgcaggac
     2881 accagccttg aggatggctg tctcgtttac cacgtccaag tcagaggggt aaactttccc
     2941 tccaatggtc ccgtgatgca gaagaagacc aagggttggg agcctaatac agagatgatg
     3001 tatccagcag atggtggtct gaggggatac actcatatgg cactgaaagt tgatggtggt
     3061 ggccatctgt cttgctcttt cgtaacaact tacaggtcaa aaaagaccgt cgggaacatc
     3121 aagatgcccg gtatccatgc cgttgatcac cgcctggaaa ggttagagga aagtgacaat
     3181 gaaatgttcg tagtacaacg cgaacacgca gttgccaagt tcgccgggct tggtggtggg
     3241 atggacgagc tgtacaaggc tagcggatcc accggtgtct agaaagcttt gaggccggcc
     3301 tgcagggatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3361 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3421 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3481 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3541 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3601 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3661 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3721 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3781 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3841 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3901 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3961 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     4021 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4081 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4141 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4201 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4261 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     4321 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     4381 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4441 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4501 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4561 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4621 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4681 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4741 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4801 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4861 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4921 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4981 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     5041 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     5101 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     5161 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5221 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     5281 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5341 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     5401 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5461 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5521 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5581 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5641 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5701 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5761 agaggcggtt tgcgtatt
//
