LOCUS       sequence-47081-e        5466 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 5466)
  AUTHORS   Young CL, Raden DL, Caplan JL, Czymmek KJ, Robinson AS
  TITLE     Cassette series designed for live-cell imaging of proteins and 
            high-resolution techniques in yeast.
  JOURNAL   Yeast. 2012 Mar;29(3-4):119-36. doi: 10.1002/yea.2895. Epub 2012 Apr
            4.
  PUBMED    22473760
REFERENCE   2  (bases 1 to 5466)
  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: "2012-03"; volume: "29(3-4):119-36. doi"; pages: " 
            10.1002/yea.2895. Epub 2012 Apr 4"
COMMENT     Sequence Label: pCY 3010-05
FEATURES             Location/Qualifiers
     source          1..5466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             87..797
                     /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
                     ICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             87..797
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(114..142)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     737..757
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        1056..1463
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             1464..2558
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3103..3122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3104..3122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3209..3226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3380..3968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3460..3479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4139..4930,4931..4999))
                     /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     4762..4781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5000..5104)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5172..5190
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5228..5250)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5350..5369
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5450..5466,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5450..5466,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggcttaaga
       61 tcggtgacgg tgctggttta attaacagta aaggagaaga acttttcact ggagttgtcc
      121 caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg
      181 gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac
      241 tacctgttcc atggccaaca cttgtcacta ctttgacttg gggtgttcaa tgctttgcta
      301 gatacccaga tcatatgaaa cagcatgact ttttcaagag tgccatgccc gaaggttatg
      361 tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca
      421 agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag
      481 atggaaacat tcttggacac aaattggaat acaacgctat ttcagataat gtatacatca
      541 ctgcagacaa acaaaagaat ggaatcaaag ctaatttcaa aattagacac aacattgaag
      601 atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
      661 tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg
      721 aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acactaggca
      781 tggatgaact atacaaatag ggcgcgccag atctctcgag gagaacttct agtatatcta
      841 catacctaat attattgcct tattaaaaat ggaatcccaa caattacatc aaaatccaca
      901 ttctcttcaa aatcaattgt cctgtacttc cttgttcatg tgtgttcaaa aacgttatat
      961 ttataggata attatactct atttctcaac aagtaattgg ttgtttggcc gagcggtcta
     1021 aggcgcctga ttcaagaaat atcttgaccg cagttaactg tgggaatact caggtatcgt
     1081 aagatgcaag agttcgaatc tcttagcaac cattattttt ttcctcaaca taacgagaac
     1141 acacaggggc gctatcgcac agaatcaaat tcgatgactg gaaatttttt gttaatttca
     1201 gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga
     1261 gagcgccgga accggctttt catatagaat agagaagcgt tcatgactaa atgcttgcat
     1321 cacaatactt gaagttgaca atattattta aggacctatt gttttttcca ataggtggtt
     1381 agcaatcgtc ttactttcta acttttctta ccttttacat ttcagcaata tatatatata
     1441 tatttcaagg atataccatt ctaatgtctg cccctaagaa gatcgtcgtt ttgccaggtg
     1501 accacgttgg tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg
     1561 ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgatg
     1621 ctacaggtgt tccacttcca gatgaggcgc tggaagcctc caagaaggct gatgccgttt
     1681 tgttaggtgc tgtgggtggt cctaaatggg gtaccggtag tgttagacct gaacaaggtt
     1741 tactaaaaat ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat
     1801 ccgactctct tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg
     1861 ttgttgtcag agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg
     1921 atggtgtcgc ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa
     1981 tggccgcttt catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag
     2041 ctaatgtttt ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg
     2101 aattccctac attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta
     2161 agaacccaac ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct
     2221 ccgatgaagc ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct
     2281 ctttgccaga caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag
     2341 atttgccaaa gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga
     2401 aattgtcatt gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt
     2461 tggatgcagg tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg
     2521 atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaaaa agattctctt tttttatgat
     2581 atttgtacat aaactttata aatgaaattc ataatagaaa cgacacgaaa ttacaaaatg
     2641 gaatatgttc atagggtaga cgaaactata tacgcaatct acatacattt atcaagaagg
     2701 agaaaaagga ggatgtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga
     2761 taaggaaaaa gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa
     2821 agttaggtgt aacagaaaat catgaaacta tgattcctaa tttatatatt ggaggatttt
     2881 ctctaaaaaa aaaaaaatac aacaaataaa aaacactcaa tgacctgacc atttgatgga
     2941 gtttaagtca ataccttctt gaaccatttc ccataatggt gaaagttccc tcaagaattt
     3001 tactctgtca gaaacggcct taacgacgta gtcgatcgag ctcgaattca tcgatgatat
     3061 cagatccact agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat
     3121 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3181 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3241 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3301 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3361 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3421 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3481 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3541 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     3601 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3721 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3781 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3841 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3901 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3961 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4021 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4081 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4741 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5101 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     5161 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     5221 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     5281 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     5341 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     5401 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     5461 actata
//
