LOCUS       sequence-9601-ex        7649 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 7649)
  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 7649)
  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: pNC862
FEATURES             Location/Qualifiers
     source          1..7649
                     /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"
     promoter        866..1307
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    866..983
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1278..1301
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1710..1937
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             2058..2768
                     /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(2085..2113)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2708..2728
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2792..2979
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2847..2871)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            3054..4254
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        3054..3397
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3398..4051
                     /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      4057..4254
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4778..4795)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4778..4794)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4787..4809)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5007..5462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5094..5113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5304..5325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5479..5498)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5598..5620
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5658..5676)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5744..5848
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5849..5917,5918..6709)
                     /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(6067..6086)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6880..7468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7369..7388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7622..7639
                     /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 cctcgaggaa
      661 ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata atcatattac
      721 atggcattac caccatatac atatccatat ctaatcttac ttatatgttg tggaaatgta
      781 aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag taatacgctt
      841 aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg acagccctcc
      901 gacggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg aaacgcagat
      961 gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct tttatggtta
     1021 tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaattaac gaatcaaatt
     1081 aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg taattaatca
     1141 gcgaagcgat gatttttgat ctattaacag atatataaat ggaaaagctg cataaccact
     1201 ttaactaata ctttcaacat tttcagtttg tattacttct tattcaaatg tcataaaagt
     1261 atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa aaaccccgga
     1321 tctgccaggc gtgtatatag cgtggatggc caggcaactt tagtgctgac acatacaggc
     1381 atatatatat gtgtgcgacg acacatgatc atatggcatg catgtgctct gtatgtatat
     1441 aaaactcttg ttttcttctt ttctctaaat attctttcct tatacattag gtcctttgta
     1501 gcataaatta ctatacttct atagacacgc aaacacaaat acacacacta aattaccgga
     1561 tcaattcgcc tgcttatctt tcttcttccg aaagtgctac ttcagaaaga gcaagaactg
     1621 tacggataag gataagtata tcttctatct actacaatac gaaaagaact ctcgaactct
     1681 ccctcccact ttactttaac taatagatta tgcagatttt cgtcaagact ttgaccggta
     1741 aaaccataac attggaagtt gaatcttccg ataccatcga caacgttaag tcgaaaattc
     1801 aagacaagga aggtatccct ccagatcaac aaagattgat ctttgccggt aagcagctag
     1861 aagacggtag aacgctgtct gattacaaca ttcagaagga gtccacctta catcttgtgc
     1921 taaggctaag aggtggtatg cacggatccg gagcttggct gttgcccgtc tcactggtga
     1981 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     2041 cattaatgca gagatccagt aaaggagaag aacttttcac tggagttgtc ccaattcttg
     2101 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
     2161 atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc
     2221 catggccaac acttgtcact actttgactt ggggtgttca atgcttttca agatacccag
     2281 atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa
     2341 gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag
     2401 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
     2461 ttcttggaca caaattggaa tacaactata tttcacacaa tgtatacatc actgcagaca
     2521 aacaaaagaa tggaatcaaa gctcatttca aaattagaca caacattgaa gatggaagcg
     2581 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
     2641 cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag
     2701 accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac
     2761 tatacaaata gggcgcgcca cttctaaata agcgaatttc ttatgattta tgatttttat
     2821 tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt
     2881 aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt
     2941 atagtatgag gtcgctctta ttgaccacac ctctaccggc agatccgcta gggataacag
     3001 ggtaatatag atctgtttag cttgcctcgt ccccgccggg tcacccggcc agcgacatgg
     3061 aggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca tgatgtgact
     3121 gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc atacattttg
     3181 atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcagacctgc gagcagggaa
     3241 acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag agaaatataa
     3301 aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa atcttgctag
     3361 gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaggaggg cttttgtaga
     3421 aagaaatacg aacgaaacga aaatcagcgt tgccatcgct ttggacaaag ctcccttacc
     3481 tgaagagtcg aattttattg atgaacttat aacttccaag catacaaacc aaaagggaga
     3541 acaagtaatc caagtagaca cgggaattgg attcttggat cacatgtatc atgcactggc
     3601 taaacatgca ggctggagct tacgacttta ctcaagaggt gatttaatca tcgatgatca
     3661 tcacactgca gaagatactg ctattgcact tggtattgca ttcaagcagg ctatgagtaa
     3721 ctttgccggc gttaaaagat ttggacatgc ttattgtcca cttgacgaag ctctttctag
     3781 aagcgtagtt gacttgtcgg gacggcccta tgctgttatc gatttgggat taaagcgtga
     3841 aaaggttggg gaattgtcct gtgaaatgat ccctcactta ctatattcct tttcggtagc
     3901 agctggaatt actttgcatg ttacctgctt atatggtagt aatgaccatc atcgtgctga
     3961 aagcgctttt aaatctctgg ctgttgccat gcgcgcggct actagtctta ctggaagttc
     4021 tgaagtccca agcacgaagg gagtgttgta aagagtactg acaataaaaa gattcttgtt
     4081 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     4141 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     4201 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     4261 attcgatact aacgctcgac ctgcaggcat gaagatggga atctcggtcg taatgatttc
     4321 tataatgacg aaaaaaaaaa aattggaaag aaaaagcttc atggccttta taaaaaggaa
     4381 ctatccaata cctcgccaga accaagtaac agtattttac ggggcacaaa tcaagaacaa
     4441 taagacagga ctgtaaagat ggacgcattg aactccaaag aacaacaaga gttccaaaaa
     4501 gtagtggaac aaaagcaaat gaaggatttc atgcgtttgt actctaatct ggtagaaaga
     4561 tgtttcacag actgtgtcaa tgacttcaca acatcaaagc taaccaataa ggaacaaaca
     4621 tgcatcatga agtgctcaga aaagttcttg aagcatagcg aacgtgtagg gcagcgtttc
     4681 caagaacaaa acgctgcctt gggacaaggc ttgggccgat aaggtgtact ggcgtatata
     4741 tatctaatta tgtatctctg gtgtcgacaa gcttggcact ggccgtcgtt ttacaacgtc
     4801 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4861 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4921 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4981 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5041 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5101 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5161 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5221 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5281 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5341 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5401 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5461 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5521 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5581 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5641 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5701 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5761 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5821 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5881 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5941 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6001 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6061 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6121 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6181 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6241 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6301 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6361 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6421 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6481 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6541 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6601 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6661 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6721 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6781 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6841 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6901 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6961 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7021 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7081 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7141 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7201 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7261 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7321 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7381 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7441 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7501 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7561 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7621 cagcgagtca gtgagcgagg aagcggaag
//
