LOCUS       sequence-164972-        7373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing a P2A peptide (porcine teschovirus-1 derived) 
            sequence that works efficiently in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   DiSanto R, Aboulhouda S, Weinberg DE
  TITLE     The fail-safe mechanism of post-transcriptional silencing of 
            unspliced HAC1 mRNA.
  JOURNAL   Elife. 2016 Oct 1;5. pii: e20069. doi: 10.7554/eLife.20069.
  PUBMED    27692069
REFERENCE   2  (bases 1 to 7373)
  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: "Elife."; 
            date: "2016-10-1"; volume: "5. pii: e20069. doi"; pages: " 
            10.7554/eLife.20069"
COMMENT     Sequence Label: pDEW8
FEATURES             Location/Qualifiers
     source          1..7373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    76..579
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /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     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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)."
     primer_bind     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2803..3446
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3340..3363
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3463..3489
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3499..4206
                     /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"
     CDS             4249..4305
                     /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             join(4306..4308,4309..5019)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5025..5041)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5026..5042)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5044..5291
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5060..5078)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5309..5328)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5310..5328)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5338..5355)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5338..5354)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5347..5369)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5495..5950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5582..5601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5792..5813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6210..6869)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(6870..7057)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(7204..7223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7323..7345
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagggtcc ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa
      121 atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt
      181 ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt
      241 cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg
      301 aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc
      361 ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt
      421 tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat
      481 ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt
      541 aaaagatacg aggcgcgtgt aagttacagg caagcgatcc gtccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctctcgagtt tatcattatc aatactcgcc
     2821 atttcaaaga atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa
     2881 aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca
     2941 gaatatataa catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata
     3001 atggagcccg ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt
     3061 gttttcttca ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag
     3121 gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt
     3181 aaatgatgac acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct
     3241 attaccttct gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc
     3301 tgaaattatt cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt
     3361 ctgtaaatct atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt
     3421 tttaaaacac cagaacttag tttcgacgga tctagaacta tgtacccata cgatgttcct
     3481 gactatgcgg gttctggtgt gtccaaagga gaggagttaa tcaaggaaaa catgagaatg
     3541 aaagttgtca tggagggctc cgttaatggt caccaattca agtgtacagg ggaaggtgaa
     3601 ggtaatcctt acatgggtac acaaactatg agaattaaag taattgaagg cggaccacta
     3661 ccatttgcat ttgacattct ggcaacgtca ttcatgtacg gatcacgaac tttcatcaag
     3721 taccctaaag gtataccaga ctttttcaag caatcttttc cagagggttt tacatgggaa
     3781 agggttacaa gatacgaaga tgggggtgtc gtcacagtta tgcaagatac ttcattagaa
     3841 gatggctgcc ttgtctatca tgtgcaagta agaggggtga attttccttc taacggacct
     3901 gtgatgcaga aaaagaccaa aggttgggaa ccaaatactg aaatgatgta cccagctgat
     3961 ggaggtttga gaggctacac acacatggcg cttaaagttg atggtggagg tcatttgtct
     4021 tgtagttttg ttaccactta tcgttctaaa aagactgttg gcaatatcaa aatgccagga
     4081 atacatgctg tagaccacag actagaaaga ctcgaagaga gcgataacga aatgttcgtt
     4141 gtacagagag agcatgccgt agccaaattt gctggcttag gcggtggtat ggatgaattg
     4201 tataagggtt caggagctat gactgtgatg acattccagg gaccaggtgc aacaaacttc
     4261 tccctcttga aacaagcagg agatgttgag gaaaatcccg ggcctatgtc aaaaggcgag
     4321 gaactattca caggtgtcgt accaatactg gtagaattgg atggcgacgt aaacgggcat
     4381 aagttttctg tgagtggtga gggtgaaggg gacgcaactt atggcaaact tacacttaag
     4441 ttcatctgca ccacagggaa gctgcctgtc ccatggccaa ctctagttac aactctcact
     4501 tacggagtac aatgtttttc tcgataccca gaccacatga aacaacatga ctttttcaaa
     4561 tctgccatgc ctgagggata tgtgcaagag agaactattt tctttaaaga tgatggtaat
     4621 tacaaaactc gtgctgaagt gaagtttgaa ggagatacac ttgttaatag aattgagtta
     4681 aagggtattg actttaagga agatggtaat atcttaggtc acaaattgga atacaactac
     4741 aattcacata acgtttacat catggcagat aaacagaaaa acggcataaa agttaatttc
     4801 aaaattagac ataatatcga agatggttcc gttcaactcg ctgatcatta tcaacaaaat
     4861 acaccaatcg gagatggacc tgttctattg ccagacaacc attacttatc tacacagagc
     4921 gccttgtcaa aagatcctaa tgaaaagaga gatcacatgg ttttgttaga atttgtcacc
     4981 gctgcgggta taacgttggg catggatgaa ttatacaagt gaatcgatac cgtcgacctc
     5041 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     5101 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     5161 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     5221 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     5281 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     5341 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     5401 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     5461 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     5521 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5581 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5641 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5701 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5761 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5821 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5881 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5941 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6001 tagatccgtc gagttcaaga gaaaaaaaaa gaaaaagcaa aaagaaaaaa ggaaagcgcg
     6061 cctcgttcag aatgacacgt atagaatgat gcattacctt gtcatcttca gtatcatact
     6121 gttcgtatac atacttactg acattcatag gtatacatat atacacatgt atatatatcg
     6181 tatgctgcag ctttaaataa tcggtgtcac tacataagaa cacctttggt ggagggaaca
     6241 tcgttggtac cattgggcga ggtggcttct cttatggcaa ccgcaagagc cttgaacgca
     6301 ctctcactac ggtgatgatc attcttgcct cgcagacaat caacgtggag ggtaattctg
     6361 ctagcctctg caaagctttc aagaaaatgc gggatcatct cgcaagagag atctcctact
     6421 ttctcccttt gcaaaccaag ttcgacaact gcgtacggcc tgttcgaaag atctaccacc
     6481 gctctggaaa gtgcctcatc caaaggcgca aatcctgatc caaacctttt tactccacgc
     6541 gccagtaggg cctctttaaa agcttgaccg agagcaatcc cgcagtcttc agtggtgtga
     6601 tggtcgtcta tgtgtaagtc accaatgcac tcaacgatta gcgaccagcc ggaatgcttg
     6661 gccagagcat gtatcatatg gtccagaaac cctatacctg tgtggacgtt aatcacttgc
     6721 gattgtgtgg cctgttctgc tactgcttct gcctcttttt ctgggaagat cgagtgctct
     6781 atcgctaggg gaccaccctt taaagagatc gcaatctgaa tcttggtttc atttgtaata
     6841 cgctttacta gggctttctg ctctgtcatc tttgccttcg tttatcttgc ctgctcattt
     6901 tttagtatat tcttcgaaga aatcacatta ctttatataa tgtataattc attatgtgat
     6961 aatgccaatc gctaagaaaa aaaaagagtc atccgctagg tggaaaaaaa aaaatgaaaa
     7021 tcattaccga ggcataaaaa aatatagagt gtactagagg aggccaagag taatagaaaa
     7081 agaaaattgc gggaaaggac tgtgttatga cttccctgac taatgccgtg ttcaaacgat
     7141 acctggcagt gactcctagc gctcaccaag ctcttaaaac gggaatttat ggtgcactct
     7201 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     7261 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     7321 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga
//
