LOCUS       sequence-356079-        4846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NatR multi-gene yeast expression vector (GFP-dropout).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4846)
  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 4846)
  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-DN5
FEATURES             Location/Qualifiers
     source          1..4846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             340..351
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             358..1071
                     /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      1083..1154
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1170..1197
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2369..2566
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    2571..3074
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(3118..3933)
                     /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(3118..3933)
                     /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     3841..3860
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3925..3944
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4158..4746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4647..4666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggccgcccc tgaattcgca tctagactga tgagacgtgg tagagccaca aacagccggt
       61 acaagcaacg atctccagga ccatctgaat catgcgcgga tgacacgaac tcacgacggc
      121 gatcacagac attaacccac agtacagaca ctgcgacaac gtggcaattc gtcgcaatac
      181 aacgtgagac cgaaagtgaa acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt
      241 atttaataat gtgtgcggca attcacattt aatttatgaa tgttttctta acatcgcggc
      301 aactcaagaa acggcaggtt cggatcttag ctactagaga aagaggagaa atactagatg
      361 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
      421 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
      481 ctgacgctga agttcatctg tactactggt aaactgccgg ttccttggcc gactctggta
      541 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
      601 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
      661 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
      721 cgcattgagc tgaaaggcat tgactttaaa gaggacggca atatcctggg ccataagctg
      781 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
      841 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
      901 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
      961 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
     1021 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactgtacaa atgaccaggc
     1081 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1141 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     1201 ataggtctca gctggaaatc tgctcgtcag tggtgctcac actgacgaat catgtacaga
     1261 tcataccgat gactgcctgg cgactcacaa ctaagcaaga cagccggaac cagcgccggc
     1321 gaacaccact gcatatatgg catatcacaa cagtccacgt ctcaagcagt tacagagatg
     1381 ttacgaacca ctagtgcact gcagtacagt ttagcttgcc tcgtccccgc cgggtcaccc
     1441 ggccagcgac atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg
     1501 ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc
     1561 atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctccagac
     1621 ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct
     1681 gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt
     1741 taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaaa aatgggtact
     1801 accttagatg atacagccta cagatacaga acatcagtcc ctggtgatgc tgaagcaatt
     1861 gaggctttag acggttcatt caccaccgac accgtcttta gagtaaccgc caccggtgat
     1921 ggatttacct taagagaagt cccagtcgac cctccattaa ctaaagtctt tccagatgat
     1981 gaatctgatg acgaaagcga cgacggagaa gatggtgacc cagattcaag aactttcgta
     2041 gcatacggtg atgacggtga tttggctggt tttgtagtcg tttcttattc aggttggaat
     2101 agaaggttga ccgttgaaga tatagaagtc gccccagagc atagaggtca tggtgtagga
     2161 agagctttga tgggtttggc tacagaattt gcaagagaga gaggagccgg tcatttatgg
     2221 ttagaagtta ctaatgttaa cgcccctgct atccatgctt atagaagaat gggtttcaca
     2281 ttatgtggtt tagatactgc tttatatgat ggaacagcat ctgacggtga acaggccttg
     2341 tatatgtcta tgccttgccc ttaaagtaac tgacaataaa aagattcttg ttttcaagaa
     2401 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     2461 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     2521 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactggagt atcacgtgct
     2581 ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa atagaaagta
     2641 aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg
     2701 gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa
     2761 ttgtttcatc ttgtctgtgt agaaaaccac acacgaaaat cctgtgattt tacattttac
     2821 ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg
     2881 taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt cttcattccg
     2941 taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat
     3001 aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt
     3061 acaggcaagc gatccgtccc gagcggccgc gtgttacaac caattaacca attctgatta
     3121 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     3181 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     3241 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     3301 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     3361 atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc
     3421 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     3481 ctgagcgagg cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     3541 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     3601 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     3661 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     3721 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     3781 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     3841 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     3901 ggagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     3961 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     4021 attttgagac acaacgtggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca
     4081 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4141 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4201 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4261 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4321 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4381 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4441 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4501 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4561 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4621 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4681 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4741 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4801 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtg
//
