LOCUS       sequence-9992-ex        7800 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 7800)
  AUTHORS   Yu RC, Pesce CG, Colman-Lerner A, Lok L, Pincus D, Serra E, Holl M, 
            Benjamin K, Gordon A, Brent R
  TITLE     Negative feedback that improves information transmission in yeast 
            signalling.
  JOURNAL   Nature. 2008 Dec 11. 456(7223):755-61.
  PUBMED    19079053
REFERENCE   2  (bases 1 to 7800)
  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: "Nature. 
            2008 Dec 11. 456(7223):755-61."
COMMENT     Sequence Label: pRY1004
FEATURES             Location/Qualifiers
     source          1..7800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             4563..5276
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4563..5276
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4593..4621)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5216..5236
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5433..5620
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5488..5512)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5760..5776)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5760..5776)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5808..5827)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5809..5827)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5834..5851)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5834..5850)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5843..5865)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5995..6450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6082..6101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6292..6313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6581..7384)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(7385..7605)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7631..7650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7750..7772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggca gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg ggggaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggccgag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctgggtaccc
     2281 ttcaagggaa cgatggtgtt ggcaatctgg atgcacagat cactgttggc caaagattgc
     2341 catctgatga tttgcgcttg ggcatttaag tcatcgtcgg cacctaaaag ttgagtcttc
     2401 atcttgtcgt cctgtgaaag ctttactaaa taatagttaa tcgccattgc ctcggttagc
     2461 ttgtaaccct ttggacccac gaaagccgga accttcttca agggaaagtc ccttgcgaac
     2521 tgttcagcgg cggcatctgg cgtaacaacc ttaacgtcca gcttcaaagc cttgacaaga
     2581 cctcttggga cccaagttct aattctgaag ttagcatata aagtaccttg agacatggcg
     2641 tgcaaaaact gtatctgatc ttgagtttct atgttctgta tgctctaata acagaaaata
     2701 caataacaag gagggaagac caatttgcgg gctgccaagc aacacaacca aaccctgctg
     2761 ataaacgaaa acaagaaaac taataaaaaa ataaataagg aactctacaa ggactacact
     2821 aatcatgtct cactcccgcg gattgaaaaa acgaaagaaa aaaacaggaa tcaacgccca
     2881 acgacctgca gcagcaagcg tgagaaattc cccgagtggg tcaccgcgag cggaagatga
     2941 aacaagcagc agtcggcttg cacaggcctt gcatgtatgc gttggtgggc cttgggcttt
     3001 acttataagc agctgcttcg ccacttgata cggttgttct ttacatctgt ttctcgctgg
     3061 gggaagttta ttaggtttag tttttccgct ttgtgctttg cgttttttta gcttatacag
     3121 aagaaacgca cttaaaaaga atcaaatcag taacaaattt tggtattgtt tctcaaagaa
     3181 gaaaatagaa agtgagaatg gccgtatcag cccgtttgag aacgactgcc gaggatacat
     3241 ccattgctaa atcaacacaa gatccaattg gtgatacaga aatcagtgta gcaaatgcta
     3301 agggcagcag cgatagcaac attaagaatt cgccaggcgg aaacagcgtt ggtcaggagt
     3361 cggagctaga gcatgtccct gaggaggatg actctggtga caaggaagca gatcatgagg
     3421 attctgagac agccactgcg aagaagagga aagcccaacc attgaagaat ccgaagaaat
     3481 cattgaagag gggcagagtc ccggcgcctt tgaatttatc ggatagcaac actaatacac
     3541 acggtggtaa tattaaggac ggcaacttgg cttcgtctaa ctctgcacat tttcctcctg
     3601 ttgctaatca aaacgtgaaa agcgcgcccg cacaagttac tcagcattcc aagttccagc
     3661 cccgtgtcca atacttggga aaggccagtt ctagacaatc tatacaagtg aataatagca
     3721 gcaatagtta tgggaaacca cacatgccct cggcgggcat catgagcgcc atgaaccctt
     3781 acatgcccat gaatcgctac ataatgtcac catattataa tccgtacggt atccctccac
     3841 ctcacatgct gaacaagccc ataatgacac cctacgtgtc gtatccatat ccaatgggac
     3901 cgcggacctc cattccttat gctatgcaag gtggcaacgc taggccctac gaagaaaatg
     3961 agtatagtgc tagcaattac agaaacaaga gggttaacga ctcatatgat tcgcctttga
     4021 gtggcaccgc ttccactggg aaaaccagac gatccgagga aggctcaaga aattctagcg
     4081 taggatcaag tgcgaatgcc ggtcctacgc aacagcgcgc ggatctacgc ccagcagata
     4141 tgatacctgc tgaagaatac cactttgaac gggatgcatt actctcggcc aacacgaaag
     4201 ccagaagcgc aagcacaagc acaagtacaa gcacaagcac aaaccgggac aggagttcat
     4261 ggcatgaggc agaacccaat aaagacgaag aagagggtac ggacttggcc atcgaggacg
     4321 gagcggtccc cactcccact tttactacgt tccagcggac ctcgcagccg cagcagcaat
     4381 cacctagtct tcttcaaggc gaaatccgac tctcatcgca tatttttgcc ttcgagttcc
     4441 ccctgagctc tagcaatgta gacaagaaaa tgtttatgag catatgtaat aaagtatgga
     4501 atgaatcaaa ggagctgaca aaaaaatcat catcacatca cagaaccgga aaagggcgcg
     4561 ccatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
     4621 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
     4681 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
     4741 ttgtcactac tttcggttat ggtttacaat gctttgctag atacccagat catatgaaac
     4801 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
     4861 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
     4921 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca
     4981 aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg
     5041 gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag
     5101 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     5161 acctgtccta tcaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
     5221 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaagcggt
     5281 cggggtgaac tcgagaagct ttggacttct tcgccagagg tttggtcaag tctccaatca
     5341 aggttgtcgg cttgtctacc ttgccagaaa tttacgaaaa gatggaaaag ggtcaaatcg
     5401 ttggtagata cgttgttgac acttctaaat aagcgaattt cttatgattt atgattttta
     5461 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     5521 taaaacgaaa attcttgttc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     5581 tatagcatga ggtcgctctt attgaccaca cctctaccgg catgccgagc aaatgcctgc
     5641 aaatcgctcc ccatttcacc caattgtaga tatgctaact ccagcaatga gttgatgaat
     5701 ctcggtgtgt attttatgtc ctcagaggac aacacctgtt gtaatcgttc ttccacacgg
     5761 atccactagt tctagagcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt
     5821 cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     5881 cccaacttaa tcgccttgca gcacatcccc ccttcgccag ctggcgtaat agcgaagagg
     5941 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc
     6001 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     6061 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     6121 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     6181 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     6241 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     6301 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     6361 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6421 attttaacaa aatattaacg tttacaattt cctgatgcgg tattttctcc ttacgcatct
     6481 gtgcggtatt tcacaccgca tagggtaata actgatataa ttaaattgaa gctctaattt
     6541 gtgagtttag tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc
     6601 ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc
     6661 ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat
     6721 catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg
     6781 gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa
     6841 agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc
     6901 cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct
     6961 ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt
     7021 gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt
     7081 tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg
     7141 cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca
     7201 catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct
     7261 tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa
     7321 atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg
     7381 acatgattta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg
     7441 ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct
     7501 ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaagaaacc
     7561 gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa ttgaaaagct gtggtatggt
     7621 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7681 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7741 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
