LOCUS       sequence-9595-ex        6249 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 6249)
  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 6249)
  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: pNC829
FEATURES             Location/Qualifiers
     source          1..6249
                     /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             658..1368
                     /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(685..713)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1308..1328
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1392..1579
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1447..1471)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1654..2854
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        1654..1997
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1998..2651
                     /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      2657..2854
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3378..3395)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3378..3394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3387..3409)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3607..4062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3694..3713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3904..3925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4079..4098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4198..4220
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4258..4276)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4344..4448
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4449..4517,4518..5309)
                     /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(4667..4686)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5480..6068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5969..5988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6222..6239
                     /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 gggatccagt
      661 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt
      721 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt
      781 acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact
      841 actttgactt ggggtgttca atgcttttca agatacccag atcatatgaa acggcatgac
      901 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat
      961 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga
     1021 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa
     1081 tacaactata tttcacacaa tgtatacatc actgcagaca aacaaaagaa tggaatcaaa
     1141 gctcatttca aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat
     1201 caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc
     1261 acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag
     1321 tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata gggcgcgcca
     1381 cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa
     1441 aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct
     1501 tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagtatgag gtcgctctta
     1561 ttgaccacac ctctaccggc agatccgcta gggataacag ggtaatatag atctgtttag
     1621 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     1681 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     1741 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     1801 aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct cacagacgcg
     1861 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     1921 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     1981 tccgaacata aacaaccatg ggtaggaggg cttttgtaga aagaaatacg aacgaaacga
     2041 aaatcagcgt tgccatcgct ttggacaaag ctcccttacc tgaagagtcg aattttattg
     2101 atgaacttat aacttccaag catacaaacc aaaagggaga acaagtaatc caagtagaca
     2161 cgggaattgg attcttggat cacatgtatc atgcactggc taaacatgca ggctggagct
     2221 tacgacttta ctcaagaggt gatttaatca tcgatgatca tcacactgca gaagatactg
     2281 ctattgcact tggtattgca ttcaagcagg ctatgagtaa ctttgccggc gttaaaagat
     2341 ttggacatgc ttattgtcca cttgacgaag ctctttctag aagcgtagtt gacttgtcgg
     2401 gacggcccta tgctgttatc gatttgggat taaagcgtga aaaggttggg gaattgtcct
     2461 gtgaaatgat ccctcactta ctatattcct tttcggtagc agctggaatt actttgcatg
     2521 ttacctgctt atatggtagt aatgaccatc atcgtgctga aagcgctttt aaatctctgg
     2581 ctgttgccat gcgcgcggct actagtctta ctggaagttc tgaagtccca agcacgaagg
     2641 gagtgttgta aagagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     2701 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     2761 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     2821 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgctcgac
     2881 ctgcaggcat gaagatggga atctcggtcg taatgatttc tataatgacg aaaaaaaaaa
     2941 aattggaaag aaaaagcttc atggccttta taaaaaggaa ctatccaata cctcgccaga
     3001 accaagtaac agtattttac ggggcacaaa tcaagaacaa taagacagga ctgtaaagat
     3061 ggacgcattg aactccaaag aacaacaaga gttccaaaaa gtagtggaac aaaagcaaat
     3121 gaaggatttc atgcgtttgt actctaatct ggtagaaaga tgtttcacag actgtgtcaa
     3181 tgacttcaca acatcaaagc taaccaataa ggaacaaaca tgcatcatga agtgctcaga
     3241 aaagttcttg aagcatagcg aacgtgtagg gcagcgtttc caagaacaaa acgctgcctt
     3301 gggacaaggc ttgggccgat aaggtgtact ggcgtatata tatctaatta tgtatctctg
     3361 gtgtcgacaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3421 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3481 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     3541 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3601 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3661 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3721 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3781 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3841 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3901 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg
     3961 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4021 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4081 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4141 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4201 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4261 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4321 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4381 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4441 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4501 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4561 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4621 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4681 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4741 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4801 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4861 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4921 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4981 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5041 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5101 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5161 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5221 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5281 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5341 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5401 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5461 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5521 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5581 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     5641 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5701 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5761 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5821 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5881 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5941 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6001 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6061 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6121 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6181 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6241 aagcggaag
//
