LOCUS       sequence-356080-        5302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HygR multi-gene yeast expression vector (GFP-dropout).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5302)
  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 5302)
  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-DN6
FEATURES             Location/Qualifiers
     source          1..5302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             381..392
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             399..1112
                     /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      1124..1195
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1211..1238
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            1489..3063
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             1833..2861
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1833..2861
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2866..3063
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    3068..3571
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(3615..4430)
                     /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(3615..4430)
                     /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     4338..4357
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4422..4441
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttctttcct gcgttatccc ctgattctgt ggataaccgt gcggccgccc ctgaattcgc
       61 atctagactg atgagacgtg gtagagccac aaacagccgg tacaagcaac gatctccagg
      121 accatctgaa tcatgcgcgg atgacacgaa ctcacgacgg cgatcacaga cattaaccca
      181 cagtacagac actgcgacaa cgtggcaatt cgtcgcaata caacgtgaga ccgaaagtga
      241 aacgtgattt catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc
      301 aattcacatt taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt
      361 tcggatctta gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagctgt
      421 tcactggtgt cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt
      481 ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct
      541 gtactactgg taaactgccg gttccttggc cgactctggt aacgacgctg acttatggtg
      601 ttcagtgctt tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca
      661 tgccggaagg ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa
      721 cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca
      781 ttgactttaa agaggacggc aatatcctgg gccataagct ggaatacaat tttaacagcc
      841 acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc
      901 gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa
      961 tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt
     1021 ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg
     1081 gcatcacgca tggtatggat gaactgtaca aatgaccagg catcaaataa aacgaaaggc
     1141 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     1201 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataggtctc agctggaaat
     1261 ctgctcgtca gtggtgctca cactgacgaa tcatgtacag atcataccga tgactgcctg
     1321 gcgactcaca actaagcaag acagccggaa ccagcgccgg cgaacaccac tgcatatatg
     1381 gcatatcaca acagtccacg tctcaagcag ttacagagat gttacgaacc actagtgcac
     1441 tgcagtacag tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc
     1501 cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc
     1561 ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc
     1621 cgcacggcgc gaagcaaaaa ttacggctcc tcgctccaga cctgcgagca gggaaacgct
     1681 cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt
     1741 taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac
     1801 agttctcaca tcacatccga acataaacaa aaatgggtaa aaagcctgaa ctcaccgcga
     1861 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt gtccgacctg atgcagctct
     1921 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     1981 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     2041 cggccgcgct cccgattccg gaagtgcttg acattgggga atttagcgag agcctgacct
     2101 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     2161 ccgctgttct gcaaccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     2221 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     2281 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2341 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     2401 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     2461 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     2521 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     2581 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     2641 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     2701 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     2761 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     2821 gaaaccgacg ccccagcact cgtccgaggg caaaggaata aagtaactga caataaaaag
     2881 attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt
     2941 taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg
     3001 aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata
     3061 ctggagtatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa
     3121 attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat
     3181 gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct
     3241 caggtattaa tgccgaattg tttcatcttg tctgtgtaga aaaccacaca cgaaaatcct
     3301 gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg
     3361 tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt
     3421 tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac
     3481 aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata
     3541 cgaggcgcgt gtaagttaca ggcaagcgat ccgtcccgag cggccgcgtg ttacaaccaa
     3601 ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     3661 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     3721 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     3781 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     3841 ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact
     3901 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     3961 ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa aggacaatta
     4021 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     4081 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     4141 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     4201 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     4261 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     4321 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     4381 gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     4441 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     4501 gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa tcgaactttt
     4561 gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5281 tttgctggcc ttttgctcac at
//
