LOCUS       sequence-9613-ex        6986 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 6986)
  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 6986)
  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: pNC955
FEATURES             Location/Qualifiers
     source          1..6986
                     /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             1095..1322
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1395..2105
                     /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(1422..1450)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2045..2065
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2129..2316
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2184..2208)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2391..3591
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        2391..2734
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2735..3388
                     /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      3394..3591
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4115..4132)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4115..4131)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4124..4146)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4344..4799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4431..4450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4641..4662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4816..4835)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4935..4957
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4995..5013)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5081..5185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5186..5254,5255..6046)
                     /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(5404..5423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6217..6805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6706..6725
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6959..6976
                     /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 ccggtacccc cctcgagtga
      661 tgaaacaaac atgaaacgtc tgtaatttga aacaaatcga gcagatctgc caggcgtgta
      721 tatagcgtgg atggccaggc aactttagtg ctgacacata caggcatata tatatgtgtg
      781 cgacgacaca tgatcatatg gcatgcatgt gctctgtatg tatataaaac tcttgttttc
      841 ttcttttctc taaatattct ttccttatac attaggtcct ttgtagcata aattactata
      901 cttctataga cacgcaaaca caaatacaca cactaaatta ccggatcaat tcgcctgctt
      961 atctttcttc ttccgaaagt gctacttcag aaagagcaag aactgtacgg ataaggataa
     1021 gtatatcttc tatctactac aatacgaaaa gaactctcga actctccctc ccactttact
     1081 ttaactaata gattatgcag attttcgtca agactttgac cggtaaaacc ataacattgg
     1141 aagttgaatc ttccgatacc atcgacaacg ttaagtcgaa aattcaagac aaggaaggta
     1201 tccctccaga tcaacaaaga ttgatctttg ccggtaagca gctagaagac ggtagaacgc
     1261 tgtctgatta caacattcag aaggagtcca ccttacatct tgtgctaagg ctaagaggtg
     1321 gttatcacgg atccggagct tggctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     1381 tggcgcccaa tacgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
     1441 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg
     1501 caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat
     1561 ggccaacact tgtcactact ttgacttggg gtgttcaatg cttttcaaga tacccagatc
     1621 atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa
     1681 ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg
     1741 atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc
     1801 ttggacacaa attggaatac aactatattt cacacaatgt atacatcact gcagacaaac
     1861 aaaagaatgg aatcaaagct catttcaaaa ttagacacaa cattgaagat ggaagcgttc
     1921 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
     1981 acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc
     2041 acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat
     2101 acaaataggg cgcgccactt ctaaataagc gaatttctta tgatttatga tttttattat
     2161 taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa
     2221 acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata
     2281 gtatgaggtc gctcttattg accacacctc taccggcaga tccgctaggg ataacagggt
     2341 aatatagatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg
     2401 cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc
     2461 gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata cattttgatg
     2521 gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg
     2581 ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag
     2641 gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat
     2701 acagttctca catcacatcc gaacataaac aaccatgggt aggagggctt ttgtagaaag
     2761 aaatacgaac gaaacgaaaa tcagcgttgc catcgctttg gacaaagctc ccttacctga
     2821 agagtcgaat tttattgatg aacttataac ttccaagcat acaaaccaaa agggagaaca
     2881 agtaatccaa gtagacacgg gaattggatt cttggatcac atgtatcatg cactggctaa
     2941 acatgcaggc tggagcttac gactttactc aagaggtgat ttaatcatcg atgatcatca
     3001 cactgcagaa gatactgcta ttgcacttgg tattgcattc aagcaggcta tgagtaactt
     3061 tgccggcgtt aaaagatttg gacatgctta ttgtccactt gacgaagctc tttctagaag
     3121 cgtagttgac ttgtcgggac ggccctatgc tgttatcgat ttgggattaa agcgtgaaaa
     3181 ggttggggaa ttgtcctgtg aaatgatccc tcacttacta tattcctttt cggtagcagc
     3241 tggaattact ttgcatgtta cctgcttata tggtagtaat gaccatcatc gtgctgaaag
     3301 cgcttttaaa tctctggctg ttgccatgcg cgcggctact agtcttactg gaagttctga
     3361 agtcccaagc acgaagggag tgttgtaaag agtactgaca ataaaaagat tcttgttttc
     3421 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     3481 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     3541 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     3601 cgatactaac gctcgacctg caggcatgaa gatgggaatc tcggtcgtaa tgatttctat
     3661 aatgacgaaa aaaaaaaaat tggaaagaaa aagcttcatg gcctttataa aaaggaacta
     3721 tccaatacct cgccagaacc aagtaacagt attttacggg gcacaaatca agaacaataa
     3781 gacaggactg taaagatgga cgcattgaac tccaaagaac aacaagagtt ccaaaaagta
     3841 gtggaacaaa agcaaatgaa ggatttcatg cgtttgtact ctaatctggt agaaagatgt
     3901 ttcacagact gtgtcaatga cttcacaaca tcaaagctaa ccaataagga acaaacatgc
     3961 atcatgaagt gctcagaaaa gttcttgaag catagcgaac gtgtagggca gcgtttccaa
     4021 gaacaaaacg ctgccttggg acaaggcttg ggccgataag gtgtactggc gtatatatat
     4081 ctaattatgt atctctggtg tcgacaagct tggcactggc cgtcgtttta caacgtcgtg
     4141 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4201 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4261 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4321 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4381 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4441 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4501 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4561 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4621 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4681 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4741 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4801 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4861 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4921 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4981 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5041 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5101 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5161 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5221 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5281 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5341 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5401 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5461 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5521 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5581 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5641 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5701 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5761 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5821 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5881 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5941 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6001 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6061 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6121 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6181 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6241 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6301 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6361 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6421 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6481 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6541 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6601 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6661 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6721 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6781 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6841 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6901 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     6961 cgagtcagtg agcgaggaag cggaag
//
