LOCUS       sequence-342853-        7285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MarkerFree plasmid for integration of pADH1-MCP-VPR into site XI-5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7285)
  AUTHORS   Jensen ED, Ferreira R, Jakociunas T, Arsovska D, Zhang J, Ding L, 
            Smith JD, David F, Nielsen J, Jensen MK, Keasling JD
  TITLE     Transcriptional reprogramming in yeast using dCas9 and combinatorial
            gRNA strategies.
  JOURNAL   Microb Cell Fact. 2017 Mar 15;16(1):46. doi: 
            10.1186/s12934-017-0664-2.
  PUBMED    28298224
REFERENCE   2  (bases 1 to 7285)
  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: "Microb Cell
            Fact."; date: "2017-03-15"; volume: "16(1):46. doi"; pages: " 
            10.1186/s12934-017-0664-2"
COMMENT     Sequence Label: pEDJ-87
FEATURES             Location/Qualifiers
     source          1..7285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             205..225
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             589..609
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             634..783
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             808..828
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1258..1614
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             1633..2202
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     terminator      2219..2408
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2425..2444)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2426..2444)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2454..2470)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2463..2485)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3028..3061)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(3531..3547)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3646..3663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3817..4405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3897..3916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4527..5318,5319..5387))
                     /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     5150..5169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5388..5479)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5521..5540
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5546..5565
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5772..5805
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(6547..6712)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6633..6657
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        join(7046..7285,1..162)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
ORIGIN
        1 ttacttgaat ttgaaataaa aaaaagtttg ctgtcttgct atcaagtata aatagacctg
       61 caattattaa tcttttgttt cctcgtcatt gttctcgttc cctttcttcc ttgtttcttt
      121 ttctgcacaa tatttcaagc tataccaagc atacaatcaa ctatctcata tacaatctgt
      181 cataaaacaa tgtctctcga gatgcccaaa aagaaaagaa aagtgggtag tatggcttct
      241 aactttactc agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca
      301 agcaacttcg ctaacgggat cgctgaatgg atcagctcta actcgcgttc acaggcttac
      361 aaagtaacct gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc
      421 gaggtgccta aaggcgcctg gcgttcgtac ttaaatatgg aactaaccat tccaattttc
      481 gccacgaatt ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga
      541 aacccgattc cctcagcaat cgcagcaaac tccggcatct acggctcccc caagaagaag
      601 aggaaggtgg aggccagcgg ttccggacgg gctgacgcat tggacgattt tgatctggat
      661 atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt
      721 gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg
      781 ctgattaact ctagaagttc cggatctccg aaaaagaaac gcaaagttgg tagccagtac
      841 ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac ctacgagaca
      901 ttcaagagca tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct
      961 agaagaatcg ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct
     1021 taccccttca ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc
     1081 cccagcggcc agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct
     1141 caggctcctg ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca
     1201 cccgtgcctg tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct
     1261 acacaggccg gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag
     1321 gatctgggag ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc
     1381 gtggacaaca gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc
     1441 accgagccca tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag
     1501 aggcctcctg atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg
     1561 tctggcgacg aggacttcag ctctatcgcc gacatggact tctccgcact gctgggtagc
     1621 ggatcgggat ctcgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc
     1681 gctattagtg acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt
     1741 catcctccag gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca
     1801 accggtccag tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg
     1861 gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt tggaggatcc cgatgaagag
     1921 acgagccagg ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa
     1981 gaggctgcaa tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat
     2041 gagctgacaa ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc
     2101 ccggaattga acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg
     2161 catatcagca caggactgtc catcttcgac acatctctgt tttgaatcgc gtgcattcat
     2221 ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt
     2281 ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt
     2341 acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac
     2401 gctcgaagat cgcgtcccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     2461 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg actagtgctg
     2521 aggcattact attggctgct tcatagtaca cccaattgcc cacaacccaa gttaatcata
     2581 agctcttgtt gtcgatgaat tgctcaaaat gtggccattc tttcaatttt gcacctctaa
     2641 aactgctccg tgggttgtga caacaacctt aagggtaatg gactttttta gaaagacaaa
     2701 tgagatgatt tcacctactg gtatatcatt tatttataca cgaccgtttt gacatttcag
     2761 agttgcacgt tggtatatcc taatattctc acaagcatca cagtgaagaa acgcaagaaa
     2821 atggaaaaaa ttcccaggtg gttgttgtct tctcttatat cgtcggtgct gtgcatactt
     2881 ggggccctgt gtgtgccgtt gttatcggtt gccttcgata gcaagcgcaa tagccaatct
     2941 aagttggtca actatggtct ttctctaagt gccggatcta tgatcattaa gacctcagcg
     3001 cggccgcaaa tttaaataaa atgaagtgaa gttcctatac tttctagaga ataggaactt
     3061 ctatagtgag tcgaataagg gcgacacaaa atttattcta aatgcataat aaatactgat
     3121 aacatcttat agtttgtatt atattttgta ttatcgttga catgtataat tttgatatca
     3181 aaaactgatt ttccctttat tattttcgag atttattttc ttaattctct ttaacaaact
     3241 agaaatattg tatatacaaa aaatcataaa taatagatga atagtttaat tataggtgtt
     3301 catcaatcga aaaagcaacg tatcttattt aaagtgcgtt gcttttttct catttataag
     3361 gttaaataat tctcatatat caagcaaagt gacaggcgcc cttaaatatt ctgacaaatg
     3421 ctctttccct aaactccccc cataaaaaaa cccgccgaag cgggttttta cgttatttgc
     3481 ggattaacga ttactcgtta tcagaaccgc ccagggggcc cgagcttaag actggccgtc
     3541 gttttacaac acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct
     3601 gcttagtttg atgcctggca gttccctact ctcgccttcc gcttcctcgc tcactgactc
     3661 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3721 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3781 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3841 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3901 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3961 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4021 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4081 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4141 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4201 tgtaggcggt gctacagagt tcttgaagtg gtgggctaac tacggctaca ctagaagaac
     4261 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4321 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4381 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4441 tcagtggaac gacgcgcgcg taactcacgt taagggattt tggtcatgag cttgcgccgt
     4501 cccgtcaagt cagcgtaatg ctctgcttac caatgcttaa tcagtgaggc acctatctca
     4561 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4621 atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga accacgctca
     4681 ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4741 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4801 agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca
     4861 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4921 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4981 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5041 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5101 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5161 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5221 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5281 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5341 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt cttccttttt
     5401 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5461 atttagaaaa ataaacaaat aggggtcagt gttacaacca attaaccaat tctgaacatt
     5521 atcgcgagcc catttatacc tgaatatggc tcataacacc ccttgtttgc ctggcggcag
     5581 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     5641 tggtagtgtg gggactcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     5701 aggctcagtc gaaagactgg gcctttcgcc cgggctaatt atggggtgtc gcccttattc
     5761 gactctatag tgaagttcct attctctaga aagtatagga acttctgaag tggggattta
     5821 aatgcggccg cgctgagggt ttaatgcgga gaagtcgttg atagcatttc cgaaggcttt
     5881 tccacattga ctgcagtatc taaaagagct ccagcagcgg tcttagtagt tcctgtgctt
     5941 gccgctttgc agtatacagg gacattggga actacggagg aaaaatcgga atgctctgag
     6001 ctcttctcaa aaccagacga actcgtctta tttctcagta aattacccaa catataaact
     6061 ctccctcttc accttctcta caaggccaca cacaagacag ttttaacagg caaatggtac
     6121 tggaggctct caccaatggg gtttaagacg ctccaaacac acgctctcct ttttttcttt
     6181 atatatcggt agcaacgaaa gctagtcgca gaacgttaca cactaaagca gatcaatcaa
     6241 tcaatcctga gaacctgctt cgttcaaacc atccactaga agaaacacgc acgtgtaccc
     6301 aaaaatagag acaaacaaac agacaaaaat aacaatgatt cctaccgact aaagtaaaac
     6361 aacactcacg taataaaaaa tcctagatac aactcacttg tattcctctc ttctttgacc
     6421 atcactaaag cttctctctt actatattcc tcaaataaaa aaaagttgca atgattactt
     6481 accaatgtgc cataaactcc gtgcaccaat taagtcctca gcgagctcgc atggaatgcg
     6541 tgcgatgagc gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac
     6601 tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta
     6661 ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc gcttatttag
     6721 aagtgtcaac aacgtatcta ccaacggaat gcgtgcgatg ggtgtacaat atggacttcc
     6781 tcttttctgg caaccaaacc catacatcgg gattcctata ataccttcgt tggtctccct
     6841 aacatgtagg tggcggaggg gagatataca atagaacaga taccagacaa gacataatgg
     6901 gctaaacaag actacaccaa ttacactgcc tcattgatgg tggtacataa cgaactaata
     6961 ctgtagccct agacttgata gccatcatca tatcgaagtt tcactaccct ttttccattt
     7021 gccatctatt gaagtaataa taggcgcatg caacttcttt tctttttttt tcttttctct
     7081 ctcccccgtt gttgtctcac catatccgca atgacaaaaa aatgatggaa gacactaaag
     7141 gaaaaaatta acgacaaaga cagcaccaac agatgtcgtt gttccagagc tgatgagggg
     7201 tatctcgaag cacacgaaac tttttccttc cttcattcac gcacactact ctctaatgag
     7261 caacggtata cggccttcct tccag
//
