LOCUS       sequence-6747-ex        5014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5014)
  AUTHORS   Sheff MA, Thorn KS
  TITLE     Optimized cassettes for fluorescent protein tagging in Saccharomyces
            cerevisiae.
  JOURNAL   Yeast 2004 Jun;21(8):661-70.
  PUBMED    15197731
REFERENCE   2  (bases 1 to 5014)
  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: "Yeast"; 
            date: "2004-06"; volume: "21(8)"; pages: "661-70"
COMMENT     Sequence Label: pKT0220
FEATURES             Location/Qualifiers
     source          1..5014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      19..206
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(74..98)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            281..1637
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        281..624
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             625..1434
                     /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(692..711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1440..1637
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1741..1760)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1742..1760)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1847..1864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2018..2606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2098..2117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2777..3568,3569..3637))
                     /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     3400..3419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3638..3742)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3810..3828
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3866..3888)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3988..4007
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4088..4106
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4088..4105
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4176..4889
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4911..5000
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
ORIGIN
        1 cgcgccactt ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt
       61 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc
      121 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc
      181 gctcttattg accacacctc taccggcaga tccgctaggg ataacagggt aatatagatc
      241 tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac
      301 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
      361 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
      421 gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac
      481 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
      541 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca
      601 catcacatcc gaacataaac aaccatgggt aaggaaaaga ctcacgtttc gaggccgcga
      661 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
      721 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
      781 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
      841 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
      901 tggttactca ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct
      961 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     1021 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     1081 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     1141 gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc
     1201 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     1261 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     1321 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     1381 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagta
     1441 ctgacaataa aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg
     1501 tagttgttct attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca
     1561 tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa
     1621 tgctggtcgc tatactgctg tcgattcgat actaacgccg ccatccagtt taaacgagct
     1681 cgaattcatc gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct
     1741 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     1801 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1861 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1921 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1981 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2041 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2101 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2161 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac
     2221 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2281 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2341 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2401 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     2461 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2521 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2581 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2641 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2701 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2761 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2821 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2881 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2941 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3001 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3061 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3121 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3181 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3241 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3301 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3361 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3421 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3481 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3541 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3601 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3661 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3721 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     3781 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     3841 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3901 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3961 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4021 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     4081 acatacgatt taggtgacac tatagaacgc ggccgccagc tgaagcttcg tacgctgcag
     4141 gtcgacggat cggtgacggt gctggtttaa ttaacatgtc taaaggtgaa gaattattca
     4201 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     4261 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     4321 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaggt tatggtttga
     4381 tgtgttttgc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     4441 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     4501 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     4561 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     4621 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     4681 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     4741 gtgatggtcc agtcttgtta ccagacaacc attacttatc ctatcaatct gccttatcca
     4801 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     4861 ttacccatgg tatggatgaa ttgtacaaac ccgggttaat taacatcttt tacccatacg
     4921 atgttcctga ctatgcgggc tatccgtatg acgtcccgga ctatgcagga tcctatccat
     4981 atgacgttcc agattacgct gctcagtgct gagg
//
