LOCUS       sequence-306513-        5706 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MarkerFree plasmid for integration of pADH1-PCP-Mxi1 into site X-4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5706)
  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 5706)
  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-22
FEATURES             Location/Qualifiers
     source          1..5706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..953)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     995..1014
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1020..1039
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1246..1279
                     /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(1880..2045)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1966..1990
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2379..2780
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             2823..2843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3264..3386
                     /codon_start=1
                     /product="transcriptional repressor domain from the mouse 
                     Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
                     /label=Mxi1
                     /translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
     terminator      3406..3595
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3612..3631)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3613..3631)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3641..3657)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3650..3672)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(4206..4239)
                     /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(4709..4725)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4824..4841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4995..5583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5075..5094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta tacctgaata
     1021 tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca
     1081 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga
     1141 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     1201 cgcccgggct aattatgggg tgtcgccctt attcgactct atagtgaagt tcctattctc
     1261 tagaaagtat aggaacttct gaagtgggga tttaaatgcg gccgcgctga gggtttaatc
     1321 ccaaagctaa gagtcccatt ttattcttct atatgtatat tttcgatact ctaaaccacc
     1381 ctacaatgta gccctatact aaatctgctc aattttcagc ttctacaagt gactcgaggc
     1441 cacgtggaaa gatccaacta ctccagcaca acgattcaat ataatcgatt gctccactca
     1501 taagaggcaa gaacaagctt caacttttgg taagccgccg tttataaaca gggaagatgt
     1561 cctttgtcaa gggaggcaca gagcatggcc aatttggcaa attgcaggtt tttctgagtg
     1621 aaaaatgaaa aagcattgta gtagagtcgg ctcactgaaa aaccggggag gacgaaaagg
     1681 tttccagcca cagttgtagt cacgtgcgcg ccatgctgac taatggcagc cgtcgttggg
     1741 cagaagagaa ttagtatggt acaggatacg ctaattgcgc tccaactacc aaggttgttg
     1801 agggaacact ggggcaatag gctgtcgcca ttcaagagca gattaagtcc tcagcgagct
     1861 cgcatggaat gcgtgcgatg agcgacctca tgctatacct gagaaagcaa cctgacctac
     1921 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat
     1981 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa
     2041 ttcgcttatt tagaagtgtc aacaacgtat ctaccaacgg aatgcgtgcg atgggtgtac
     2101 aatatggact tcctcttttc tggcaaccaa acccatacat cgggattcct ataatacctt
     2161 cgttggtctc cctaacatgt aggtggcgga ggggagatat acaatagaac agataccaga
     2221 caagacataa tgggctaaac aagactacac caattacact gcctcattga tggtggtaca
     2281 taacgaacta atactgtagc cctagacttg atagccatca tcatatcgaa gtttcactac
     2341 cctttttcca tttgccatct attgaagtaa taataggcgc atgcaacttc ttttcttttt
     2401 ttttcttttc tctctccccc gttgttgtct caccatatcc gcaatgacaa aaaaatgatg
     2461 gaagacacta aaggaaaaaa ttaacgacaa agacagcacc aacagatgtc gttgttccag
     2521 agctgatgag gggtatctcg aagcacacga aactttttcc ttccttcatt cacgcacact
     2581 actctctaat gagcaacggt atacggcctt ccttccagtt acttgaattt gaaataaaaa
     2641 aaagtttgct gtcttgctat caagtataaa tagacctgca attattaatc ttttgtttcc
     2701 tcgtcattgt tctcgttccc tttcttcctt gtttcttttt ctgcacaata tttcaagcta
     2761 taccaagcat acaatcaact atctcatata caatctgtca taaaacaatg tctctcgaga
     2821 tgcccaaaaa gaaaagaaaa gtgggtagtt ccaaaaccat cgttctttcg gtcggcgagg
     2881 ctactcgcac tctgactgag atccagtcca ccgcagaccg tcagatcttc gaagagaagg
     2941 tcgggcctct ggtgggtcgg ctgcgcctca cggcttcgct ccgtcaaaac ggagccaaga
     3001 ccgcgtatcg cgtcaaccta aaactggatc aggcggacgt cgttgattcc ggacttccga
     3061 aagtgcgcta cactcaggta tggtcgcacg acgtgacaat cgttgcgaat agcaccgagg
     3121 cctcgcgcaa atcgttgtac gatttgacca agtccctcgt cgcgacctcg caggtcgaag
     3181 atcttgtcgt caaccttgtg ccgctgggcc gtggtagcgg atcgggatct ggtagttcca
     3241 agcttggcgg cagcggcggc agcatggaac gtgtgagaat gattaatgtg caaaggctgt
     3301 tagaagccgc agagttttta gaaagaagag aaagagaatg cgaacacggg tatgccagtt
     3361 ctttccctag catgccctct cccagaggct aaatcgcgtg cattcatccg ctctaaccga
     3421 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     3481 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     3541 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaagatcgc
     3601 gtcccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3661 gtcgtgactg ggaaaaccct ggcgttaccc ctgcaggact agtgctgagg cattaataca
     3721 tgggaagatt cgcttttttt ttttgaatta caatagtatg tctgatgtct gcaagaagta
     3781 acaggcgtgt gcacaagaat acgtgtgtgt gcgtaagcgt atgcactggt ggcataactt
     3841 atctaagaag tatatatcac tgacatagaa atgtagatat acaggtattt ttctcgataa
     3901 tcgataaaaa tctcgtcgcg ctgaaccaaa cttggtggtt acggagagtt tttctctcat
     3961 cattactgtc tttcgcattg atttcccctt tgaccgataa aatcccttgg attcataaga
     4021 ttaaacaaag aggtgatcaa agagaaccct gtgaaagttt atgtttataa ccgggcataa
     4081 agtgaactag acactttcaa gaagccaacc aaagcatgag taacgaagct taccagcatg
     4141 atcataccgt aaatcctcac cagattaaga cctcagcgcg gccgcaaatt taaataaaat
     4201 gaagtgaagt tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc
     4261 gacacaaaat ttattctaaa tgcataataa atactgataa catcttatag tttgtattat
     4321 attttgtatt atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta
     4381 ttttcgagat ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa
     4441 atcataaata atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta
     4501 tcttatttaa agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca
     4561 agcaaagtga caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca
     4621 taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc
     4681 agaaccgccc agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt
     4741 tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt
     4801 tccctactct cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5161 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5401 ttgaagtggt gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta
     5641 actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct
     5701 ctgctt
//
