LOCUS       sequence-9599-ex        6357 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 6357)
  AUTHORS   Hackett EA, Esch RK, Maleri S, Errede B
  TITLE     A family of destabilized cyan fluorescent proteins as 
            transcriptional reporters in S. cerevisiae.
  JOURNAL   Yeast. 2006 Apr 15. 23(5):333-49.
  PUBMED    16598699
REFERENCE   2  (bases 1 to 6357)
  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. 2006
            Apr 15. 23(5):333-49."
COMMENT     Sequence Label: pNC860
FEATURES             Location/Qualifiers
     source          1..6357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             766..1476
                     /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
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(793..821)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1416..1436
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1500..1687
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1555..1579)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1762..2962
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        1762..2105
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2106..2759
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHTNQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMSNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2765..2962
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3486..3503)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3486..3502)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3495..3517)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3715..4170
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3802..3821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4012..4033
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4187..4206)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4306..4328
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4366..4384)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4452..4556
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4557..4625,4626..5417)
                     /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     complement(4775..4794)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5588..6176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6077..6096
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6330..6347
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgagc
      241 tctggagaat atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc
      301 ggctttattg ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg
      361 acacccggtg tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg
      421 gatgatgtgg tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag
      481 ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg
      541 agaagatgcg gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac
      601 tcacaaatta gagcttcaat ttaattatat cagttattac ccggtacccg gggatccgga
      661 gcttggctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
      721 accgcctctc cccgcgcgtt ggccgattca ttaatgcaga gatccagtaa aggagaagaa
      781 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
      841 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
      901 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttgacttgg
      961 ggtgttcaat gcttttcaag atacccagat catatgaaac ggcatgactt tttcaagagt
     1021 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     1081 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     1141 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactatatt
     1201 tcacacaatg tatacatcac tgcagacaaa caaaagaatg gaatcaaagc tcatttcaaa
     1261 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact
     1321 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc
     1381 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct
     1441 gctgggatta cacatggcat ggatgaacta tacaaatagg gcgcgccact tctaaataag
     1501 cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat
     1561 acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt
     1621 tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct
     1681 ctaccggcag atccgctagg gataacaggg taatatagat ctgtttagct tgcctcgtcc
     1741 ccgccgggtc acccggccag cgacatggag gcccagaata ccctccttga cagtcttgac
     1801 gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat
     1861 gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc
     1921 tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc
     1981 cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt
     2041 catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa
     2101 caaccatggg taggagggct tttgtagaaa gaaatacgaa cgaaacgaaa atcagcgttg
     2161 ccatcgcttt ggacaaagct cccttacctg aagagtcgaa ttttattgat gaacttataa
     2221 cttccaagca tacaaaccaa aagggagaac aagtaatcca agtagacacg ggaattggat
     2281 tcttggatca catgtatcat gcactggcta aacatgcagg ctggagctta cgactttact
     2341 caagaggtga tttaatcatc gatgatcatc acactgcaga agatactgct attgcacttg
     2401 gtattgcatt caagcaggct atgagtaact ttgccggcgt taaaagattt ggacatgctt
     2461 attgtccact tgacgaagct ctttctagaa gcgtagttga cttgtcggga cggccctatg
     2521 ctgttatcga tttgggatta aagcgtgaaa aggttgggga attgtcctgt gaaatgatcc
     2581 ctcacttact atattccttt tcggtagcag ctggaattac tttgcatgtt acctgcttat
     2641 atggtagtaa tgaccatcat cgtgctgaaa gcgcttttaa atctctggct gttgccatgc
     2701 gcgcggctac tagtcttact ggaagttctg aagtcccaag cacgaaggga gtgttgtaaa
     2761 gagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta
     2821 tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga
     2881 catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat
     2941 gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgctcgacct gcaggcatga
     3001 agatgggaat ctcggtcgta atgatttcta taatgacgaa aaaaaaaaaa ttggaaagaa
     3061 aaagcttcat ggcctttata aaaaggaact atccaatacc tcgccagaac caagtaacag
     3121 tattttacgg ggcacaaatc aagaacaata agacaggact gtaaagatgg acgcattgaa
     3181 ctccaaagaa caacaagagt tccaaaaagt agtggaacaa aagcaaatga aggatttcat
     3241 gcgtttgtac tctaatctgg tagaaagatg tttcacagac tgtgtcaatg acttcacaac
     3301 atcaaagcta accaataagg aacaaacatg catcatgaag tgctcagaaa agttcttgaa
     3361 gcatagcgaa cgtgtagggc agcgtttcca agaacaaaac gctgccttgg gacaaggctt
     3421 gggccgataa ggtgtactgg cgtatatata tctaattatg tatctctggt gtcgacaagc
     3481 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     3541 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     3601 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     3661 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     3721 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3781 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3841 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3901 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     3961 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4021 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     4081 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4141 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4201 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4261 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4321 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4381 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4441 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4501 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4561 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4621 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4681 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4741 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4801 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4861 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4921 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4981 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5041 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5101 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5161 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5221 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5281 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5341 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5401 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5461 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5521 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5581 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5641 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5701 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5761 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5821 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5881 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5941 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6001 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6061 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6121 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6181 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6241 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6301 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
