LOCUS       sequence-356078-        5094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KanR multi-gene yeast expression vector (GFP-dropout).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5094)
  AUTHORS   Novarina D, Koutsoumpa A, Milias-Argeitis A
  TITLE     A user-friendly and streamlined protocol for CRISPR/Cas9 genome 
            editing in budding yeast.
  JOURNAL   STAR Protoc. 2022 Jun 10;3(2):101358. doi: 
            10.1016/j.xpro.2022.101358. eCollection 2022 Jun 17.
  PUBMED    35712010
REFERENCE   2  (bases 1 to 5094)
  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: "STAR 
            Protoc."; date: "2022-06-10"; volume: "3(2):101358. doi"; pages: " 
            10.1016/j.xpro.2022.101358. eCollection 2022 Jun 17"
COMMENT     Sequence Label: pYTK-DN4
FEATURES             Location/Qualifiers
     source          1..5094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             357..368
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             375..1088
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1100..1171
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1187..1214
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            1465..2820
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             1809..2618
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      2623..2820
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    2825..3328
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(3372..4187)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3372..4187)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4179..4198
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4412..5000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4901..4920
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtgcgg ccgcccctga attcgcatct agactgatga gacgtggtag
       61 agccacaaac agccggtaca agcaacgatc tccaggacca tctgaatcat gcgcggatga
      121 cacgaactca cgacggcgat cacagacatt aacccacagt acagacactg cgacaacgtg
      181 gcaattcgtc gcaatacaac gtgagaccga aagtgaaacg tgatttcatg cgtcattttg
      241 aacattttgt aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt
      301 tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag
      361 aggagaaata ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg
      421 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
      481 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc
      541 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
      601 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
      661 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
      721 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata
      781 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
      841 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
      901 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
      961 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg
     1021 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     1081 tgtacaaatg accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1141 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     1201 tgggcctttc tgcgtttata ggtctcagct ggaaatctgc tcgtcagtgg tgctcacact
     1261 gacgaatcat gtacagatca taccgatgac tgcctggcga ctcacaacta agcaagacag
     1321 ccggaaccag cgccggcgaa caccactgca tatatggcat atcacaacag tccacgtctc
     1381 aagcagttac agagatgtta cgaaccacta gtgcactgca gtacagttta gcttgcctcg
     1441 tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt
     1501 gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc
     1561 catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac
     1621 ggctcctcgc tccagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt
     1681 ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc
     1741 tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat
     1801 aaacaaaaat gggtaaggaa aagactcacg tttcgaggcc gcgattaaat tccaacatgg
     1861 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     1921 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     1981 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     2041 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     2101 cgatccccgg caaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     2161 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     2221 cttttaacag cgatcgcgta tttcgtctgg ctcaggcgca atcacgaatg aataacggtt
     2281 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     2341 aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     2401 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     2461 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     2521 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     2581 aattgcagtt tcatttgatg ctcgatgagt ttttctaaag taactgacaa taaaaagatt
     2641 cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa
     2701 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     2761 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     2821 gagtatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt
     2881 aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta
     2941 aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag
     3001 gtattaatgc cgaattgttt catcttgtct gtgtagaaaa ccacacacga aaatcctgtg
     3061 attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct
     3121 aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt
     3181 ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa
     3241 acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga
     3301 ggcgcgtgta agttacaggc aagcgatccg tcccgagcgg ccgcgtgtta caaccaatta
     3361 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3421 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3481 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3541 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     3601 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     3661 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     3721 attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa
     3781 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3841 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3901 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3961 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     4021 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     4081 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     4141 tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta
     4201 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     4261 atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct
     4321 gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4381 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4441 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4501 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4561 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4621 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4681 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4741 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4801 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4861 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4921 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4981 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5041 gctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctga
//
