LOCUS       sequence-364606-        5994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven red fluorescent protein mRuby2 
            fused to a ""self-cleaving"" peptide p2a (PhCMV-p2a_mRuby2-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5994)
  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 5994)
  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: pTS2389-Tier1-PhCMV-p2a_mRuby
FEATURES             Location/Qualifiers
     source          1..5994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     670..689
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        670..688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             750..806
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             825..1532
                     /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(1587..1604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1593..1817
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1863..2291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1950..1969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2160..2181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2300..2320)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2305..2634
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2485..2620
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2547..2566
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2701..3495
                     /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(2755..2774)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3365..3384
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3669..3790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3706..3725)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3760..3779
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3839..3855)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3839..3855)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3852..3874)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3863..3879
                     /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(3887..3893,3894..3911,3912..3917))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3932..3953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4070..4087)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4241..4829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4321..4340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5000..5791,5792..5860))
                     /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     5623..5642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5861..5965)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc cacgcgtggt accctcgagt tgacattgat tattgagtag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
      661 tatcgaaatt aatacgactc actataggga gacccaagct gttcgaagcg gaattcacca
      721 tgactagtgg tggttctggt ggaagcggag ctactaactt cagcctgctg aagcaggctg
      781 gtgacgtgga ggagaaccct ggacctgcta gtggtggttc tggtgtgtct aagggcgaag
      841 agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga aggttcggtc aacggccacc
      901 aattcaaatg cacaggtgaa ggagaaggca atccgtacat gggaactcaa accatgagga
      961 tcaaagtcat cgagggagga cccctgccat ttgcctttga cattcttgcc acgtcgttca
     1021 tgtatggcag ccgtactttt atcaagtacc cgaaaggcat tcctgatttc tttaaacagt
     1081 cctttcctga gggttttact tgggaaagag ttacgagata cgaagatggt ggagtcgtca
     1141 ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt ttaccacgtc caagtcagag
     1201 gggtaaactt tccctccaat ggtcccgtga tgcagaagaa gaccaagggt tgggagccta
     1261 atacagagat gatgtatcca gcagatggtg gtctgagggg atacactcat atggcactga
     1321 aagttgatgg tggtggccat ctgtcttgct ctttcgtaac aacttacagg tcaaaaaaga
     1381 ccgtcgggaa catcaagatg cccggtatcc atgccgttga tcaccgcctg gaaaggttag
     1441 aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca cgcagttgcc aagttcgccg
     1501 ggcttggtgg tgggatggac gagctgtaca aggctagcgg atccaccggt gtctagaaag
     1561 ctttgaggcc ggcctgcagg gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1621 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1681 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1741 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1801 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1861 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1921 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1981 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2041 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2101 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2161 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2221 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2281 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2341 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2401 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2461 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2521 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2581 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2641 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     2701 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2761 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2821 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     2881 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     2941 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3001 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3061 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3121 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3181 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3241 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3301 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3361 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3421 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3481 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3541 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3601 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3661 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3721 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3781 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3841 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3901 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3961 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4021 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4081 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4141 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4201 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4261 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4321 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4381 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4441 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4501 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4561 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4621 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4681 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4741 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4801 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4861 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4921 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4981 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5041 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5101 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5161 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5221 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5281 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5341 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5401 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5461 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5521 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5581 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5641 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5701 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5761 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5821 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5881 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5941 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacg
//
