LOCUS       sequence-71981-e        8347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning Saccharomyces cerevisiae expression vector with GAL1 
            promoter and N-terminal 10x His tag, GFP and 3C protease cleavage 
            site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8347)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 8347)
  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: 
            "Biochemistry."; date: "2011-04-19"; volume: "50(15)"; pages: 
            "3272-8. Epub 2011 Mar 25"
COMMENT     Sequence Label: pYEXNHG3
FEATURES             Location/Qualifiers
     source          1..8347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      596..1476
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(1545..2000)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     1894..1913
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2019..2460
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2019..2136
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2431..2454
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     2492..2511
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2492..2510
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2533..2562
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2563..3273
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3280..3303
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(3352..3657)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3454..3473)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3999..4658)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4573..4592
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4712..4718,4719..4736,4737..4742))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4767..4891)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4910..4930)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4934..4954)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5008..5049
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5059..5076
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5110..5357
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5126..5144)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5434..5451)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5605..6193)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5685..5704)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6364..7155,7156..7224))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             complement(7320..8123)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(8124..8344)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
ORIGIN
        1 ctagcttatc gatgataagc tgtcaaagat gagaattaat tccacggact atagactata
       61 ctagatactc cgtctactgt acgatacact tccgctcagg tccttgtcct ttaacgaggc
      121 cttaccactc ttttgttact ctattgatcc agctcagcaa aggcagtgtg atctaagatt
      181 ctatcttcgc gatgtagtaa aactagctag accgagaaag agactagaaa tgcaaaaggc
      241 acttctacaa tggctgccat cattattatc cgatgtgacg ctgcagcttc tcaatgatat
      301 tcgaatacgc tttgaggaga tacagcctaa tatccgacaa actgttttac agatttacga
      361 tcgtacttgt tacccatcat tgaattttga acatccgaac ctgggagttt tccctgaaac
      421 agatagtata tttgaacctg tataataata tatagtctag cgctttacgg aagacaatgt
      481 atgtatttcg gttcctggag aaactattgc atctattgca taggtaatct tgcacgtcgc
      541 atccccggtt cattttctgc gtttccatct tgcacttcaa tagcatatct ttgttaacga
      601 agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac
      661 aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca
      721 acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgacgag agcgctaatt
      781 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta
      841 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc
      901 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca
      961 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg
     1021 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc
     1081 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt
     1141 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa
     1201 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt
     1261 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga
     1321 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag
     1381 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata
     1441 cttttgagca atgtttgtgg aagcggtatt cgcaatggga agctccaccc cggttgataa
     1501 tcagaaaagc cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa
     1561 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta acgaatagcc
     1621 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     1681 tccagtttcc aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     1741 aagggtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     1801 gtcgaggtgc cgtaaagcag taaatcggaa gggtaaacgg atgcccccat ttagagcttg
     1861 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggggc
     1921 tagggcggtg ggaagtgtag gggtcacgct gggcgtaacc accacacccg ccgcgcttaa
     1981 tggggcgcta cagggcgcgt ggggatgatc cactagtacg gattagaagc cgccgagcgg
     2041 gtgacagccc tccgaaggaa gactctcctc cgtgcgtcct cgtcttcacc ggtcgcgttc
     2101 ctgaaacgca gatgtgcctc gcgccgcact gctccgaaca ataaagattc tacaatacta
     2161 gcttttatgg ttatgaagag gaaaaattgg cagtaacctg gccccacaaa ccttcaaatg
     2221 aacgaatcaa attaacaacc ataggatgat aatgcgatta gttttttagc cttatttctg
     2281 gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata aatgcaaaaa
     2341 ctgcataacc actttaacta atactttcaa cattttcggt ttgtattact tcttattcaa
     2401 atgtaataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag
     2461 aaaaaacccc ggatcggact actagcagct gtaatacgac tcactatagg gaatattaag
     2521 cttggtacca tgcaccatca tcatcaccat catcatcatc attctaaagg agaagaatta
     2581 ttcactggtg ttgtcccaat tttggttgaa ttagatggtg atgttaatgg tcacaaattt
     2641 tctgtctccg gtgaaggtga aggtgatgct acttacggta aattgacctt aaaatttatt
     2701 tgtactactg gtaaattgcc agttccatgg ccaaccttag tcactacttt cggttatggt
     2761 gttcaatgtt ttgcgagata cccagatcat atgaaacaac atgacttttt caagtctgcc
     2821 atgccagaag gttatgttca agaaagaact atttttttca aagatgacgg taactacaag
     2881 accagagctg aagtcaagtt tgaaggtgat accttagtta atagaatcga attaaaaggt
     2941 attgatttta aagaagatgg taacatttta ggtcacaaat tggaatacaa ctataactct
     3001 cacaatgttt acatcatggc tgacaaacaa aagaatggta tcaaagttaa cttcaaaatt
     3061 agacacaaca ttgaagatgg ttctgttcaa ttagctgacc attatcaaca aaatactcca
     3121 attggtgatg gtccagtctt gttaccagac aaccattact tatccactca atctgcctta
     3181 tccaaagatc caaacgaaaa gagagaccac atggtcttgt tagaatttgt tactgctgct
     3241 ggtattaccc atggtatgga tgaattgtac aaaactagtt tagaagtttt gtttcaaggt
     3301 ccaagtagaa gagcgagctg cagactggct gtgtataagg gagcctgaca tttatattcc
     3361 ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac
     3421 ttcttccccg ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct
     3481 ttcatccccg atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg
     3541 tgcactggcc agggggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac
     3601 atccacaaac agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc
     3661 accagcccct gttctcgtca gcaaaagagc cgttcatttc aataaaccgg gcgacctcag
     3721 ccatcccttc ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct
     3781 gcatggttgt gcttaccaga ccggagatat tgacatcata tatgccttga gcaactgata
     3841 gctgtcgctg tcaactgtca ctgtaatacg ctgcttcata gcatacctct ttttgacata
     3901 cttcgggtat acatatcagt atatattctt ataccgcaaa aatcagcgcg caaatacgca
     3961 tactgttatc tggcttttag taagccggat ccacgcgttt acgccccgcc ctgccactca
     4021 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     4081 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     4141 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     4201 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     4261 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     4321 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     4381 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
     4441 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     4501 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     4561 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     4621 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     4681 aatctcgacg gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatgc ggccgccata gtgactggat atgttgtgtt ttacagtatt
     4801 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     4861 tttctcgttc agcttttttg tacaaacttg tgatatcgaa ttcgctagct acctttctct
     4921 tcttttttgg atctaccttt ctcttctttt ttggggatcc actagttcag ctcttctgca
     4981 taataactat ctagagggcc cttcgaaggt aagcctatcc ctaaccctct cctcggtctc
     5041 gattctacgc gtaccggtca tcatcaccat caccattgag tttaaacccg ctgatcctag
     5101 agggccgcat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     5161 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     5221 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     5281 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     5341 cgaaggcttt aatttgcaag ctgcggccct gcattaatga atcggccaac gcgcggggag
     5401 aggcggtttg cgtattgggc gctattccgc ttcctcgctc actgactcgc tgcgctcggt
     5461 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5521 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagc ccaggaaccg
     5581 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5641 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5701 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5761 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5821 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5881 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5941 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6001 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     6061 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6121 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6181 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6241 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6301 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6361 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6421 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagcgct taccatctgg
     6481 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6541 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6601 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6661 caacgttgtt ggcattgcta caggcatcgt ggtgtcactc tcgtcgtttg gtatggcttc
     6721 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6781 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6841 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6901 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6961 ttgctcttgc ccggcgtcaa tacgggataa tagtgtatca catagcagaa ctttaaaagt
     7021 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7081 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7141 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7201 gacacggaaa tgttgaatac tcatactctt cctttttcaa tgggtaataa ctgatataat
     7261 taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt
     7321 tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca
     7381 ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca
     7441 gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg
     7501 tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc
     7561 atgtctcttt gagcaataaa gccgataaca aaatctttgt cgcttttcgc aatgtcaaca
     7621 gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc
     7681 aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata
     7741 cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca
     7801 cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag
     7861 agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa
     7921 aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca
     7981 actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc
     8041 ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt
     8101 tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc
     8161 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat
     8221 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca
     8281 aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaat
     8341 tgaaaag
//
