LOCUS       sequence-358185-        8026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT compatible vector for C-terminal tagging with GFP followed by
            the CL1 degron for constitutive gene expression knockdown..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8026)
  AUTHORS   Shively CA, Dong F, Mitra RD
  TITLE     A Suite of New Strain Construction Vectors for Gene Expression 
            Knockdown in Budding Yeast.
  JOURNAL   ACS Synth Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547.
  PUBMED    36650116
REFERENCE   2  (bases 1 to 8026)
  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: "ACS Synth 
            Biol. 2023 Jan 17. doi: 10.1021/acssynbio.2c00547."
COMMENT     Sequence Label: C-SWAT GFP CL1
FEATURES             Location/Qualifiers
     source          1..8026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             440..1774
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNG
                     FGGVLSFGVKDLPNADKETDPFELSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
     rep_origin      complement(1879..2334)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2016..2037)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2228..2247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2460..2482
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2474..2491
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2475..2491
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2501..2520
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2501..2519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2544..2560
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2545..2561
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2676..3386
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2703..2731)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3326..3346
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     gene            3691..5047
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        3691..4034
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4035..4844
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4102..4121)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4850..5047
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5253..5273)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5253..5271)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5292..5308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5292..5308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5305..5327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5316..5332
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5340..5346,5347..5364,5365..5370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5385..5406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5523..5540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5694..6282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5774..5793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6453..7244,7245..7313))
                     /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     7076..7095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7314..7418)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7455..7958
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7999..8017
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accacgcttt tcaattcaat tcatcaccat cctcatgaaa actgtgtaac ataataaccg
      241 aagtgtcgaa aaggtggcac cttgtccaat tgaacacgct cgatgaaaaa aataagatat
      301 atataaggtt aagtaaagcg tctgttagaa aggaagtttt tcctttttct tgctctcttg
      361 tcttttcatc tactatttcc ttcgtgtaat acagggtcgt cagatacata gatacaattc
      421 tattaccccc atccatacaa tgccatctca tttcgatact gttcaactac acgccggcca
      481 agagaaccct ggtgacaatg ctcacagatc cagagctgta ccaatttacg ccaccacttc
      541 ttatgttttc gaaaactcta agcatggttc gcaattgttt ggtctagaag ttccaggtta
      601 cgtctattcc cgtttccaaa acccaaccag taatgttttg gaagaaagaa ttgctgcctt
      661 agaaggtggt gctgctgctt tggctgtttc ctccggtcaa gccgctcaaa cccttgccat
      721 ccaaggtttg gcacacactg gtgacaacat cgtttccact tcttacttat acggtggtac
      781 ttataaccag ttcaaaatct cgttcaaaag atttggtatc gaggctagat ttgttgaagg
      841 tgacaatcca gaagaattcg aaaaggtctt tgatgaaaga accaaggctg tttatttgga
      901 aaccattggt aatccaaagt acaatgttcc ggattttgaa aaaattgttg caattgctca
      961 caaacacggt attccagttg tcgttgacaa cacatttggt gccggtggtt acttctgtca
     1021 gccaattaaa tacggtgctg atattgtaac acattctgct accaaatgga ttggtggtca
     1081 tggtactact atcggtggta ttattgttga ctctggtaag ttcccatgga aggactaccc
     1141 agaaaagttc cctcaattct ctcaacctgc cgaaggatat cacggtacta tctacaatga
     1201 agcctacggt aacttggcat acatcgttca tgttagaact gaactattaa gagatttggg
     1261 tccattgatg aacccatttg cctctttctt gctactacaa ggtgttgaaa cattatcttt
     1321 gagagctgaa agacacggtg aaaatgcatt gaagttagcc aaatggttag aacaatcccc
     1381 atacgtatct tgggtttcat accctggttt agcatctcat tctcatcatg aaaatgctaa
     1441 gaagtatcta tctaacggtt tcggtggtgt cttatctttc ggtgtaaaag acttaccaaa
     1501 tgccgacaag gaaactgacc cattcgaact ttctggtgct caagttgttg acaatttaaa
     1561 gcttgcctct aacttggcca atgttggtga tgccaagacc ttagtcattg ctccatactt
     1621 cactacccac aaacaattaa atgacaaaga aaagttggca tctggtgtta ccaaggactt
     1681 aattcgtgtc tctgttggta tcgaatttat tgatgacatt attgcagact tccagcaatc
     1741 ttttgaaact gttttcgctg gccaaaaacc atgagtgtgc gtgatgagtt gtaaaattat
     1801 gtataaacct actttctctc acaagtactg cggtgtgaaa taccgcacag atgcgtaagg
     1861 agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     1921 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     1981 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2041 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2101 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2161 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2221 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2281 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc
     2341 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     2401 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     2461 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg
     2521 cgaattgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt
     2581 cctgcagcta tattaccctg ttatccctac gtacgctgca ggtcgacggt ggcggttctg
     2641 gcggtggcgg atcccggatc cccgggttaa ttaacagtaa aggagaagaa cttttcactg
     2701 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     2761 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     2821 ctggaaaact acctgttcca tggccaacac ttgtcactac tttcacttat ggtgttcaat
     2881 gcttttcaag atacccagat catatgaaac ggcatgactt tttcaagagt gccatgcccg
     2941 aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg
     3001 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
     3061 ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac tcacacaatg
     3121 tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa attagacaca
     3181 acattgaaga tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg
     3241 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag
     3301 atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta
     3361 cacatggcat ggatgaacta tacaaaggtg gttctggttc tgcttgtaaa aattggtttt
     3421 cttctttgtc tcattttgtt attcatttgt agagttcttc tttgagatat cgattgaacg
     3481 agctcgaatt catcgatcta tattaccctg ttatccctag cgtaactatg ccaaatgaca
     3541 ataagacacc caataggtcc agcactccca agtttactaa aaaaccagta accccgaatg
     3601 ataaaattcc tgaaagacgg atccccgggt taattaaggc gcgccagatc tgtttagctt
     3661 gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac
     3721 agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata
     3781 catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa
     3841 aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg
     3901 aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg
     3961 ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc
     4021 gaacataaac aaccatgggt aaggaaaaga ctcacgtttc gaggccgcga ttaaattcca
     4081 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     4141 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     4201 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     4261 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     4321 ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     4381 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     4441 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     4501 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     4561 tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     4621 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4681 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4741 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4801 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagta ctgacaataa
     4861 aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct
     4921 attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga
     4981 tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc
     5041 tatactgctg tcgattcgat actaacgccg ccatccagtt taaacgagct cgaattctga
     5101 tggaaaaaga ttttcaagtg aataaggaga taaaaccgta ttgacttttt atcattctac
     5161 aaaagaccag taacgaagtg taccctgcta tattaccctg ttatccctag cgtaactcgg
     5221 ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     5281 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5341 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5401 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5461 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5521 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5581 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5641 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7321 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7381 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7441 cctgggtcct tttcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa
     7501 tatataaatt aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc
     7561 cgaagatgta aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc
     7621 ctgctctcag gtattaatgc cgaattgttt catcttgtct gtgtagaaga ccacacacga
     7681 aaatcctgtg attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct
     7741 tttcttgtct aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt
     7801 gtttattttt ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc
     7861 caaatacaaa acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta
     7921 aaagatacga ggcgcgtgta agttacaggc aagcgatccg tctaagaaac cattattatc
     7981 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
