LOCUS       sequence-7814-ex        8016 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 8016)
  AUTHORS   Alberti S, Gitler AD, Lindquist S
  TITLE     A suite of Gateway cloning vectors for high-throughput genetic 
            analysis in Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2007 Oct . 24(10):913-9.
  PUBMED    17583893
REFERENCE   2  (bases 1 to 8016)
  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. 2007
            Oct . 24(10):913-9."
COMMENT     Sequence Label: pAG424GAL-ccdB
FEATURES             Location/Qualifiers
     source          1..8016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(3486..3491,3492..3509,3510..3516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /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     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3627..4068
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3627..3744
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4039..4062
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     4083..4099
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4083..4099
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    4107..4231
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4256..4261,4262..4279,4280..4286)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4340..4999
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4406..4425)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5344..5649
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     5528..5547
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5690..5814)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5847..5863)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5848..5864)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5866..6113
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5882..5900)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6131..6150)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6132..6150)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6160..6177)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6160..6176)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6169..6191)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6321..6776
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6408..6427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6618..6639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6875..7549)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(7550..7830)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7847..7866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7966..7988
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaatatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct ctagtacgga ttagaagccg ccgagcgggc gacagccctc
     3661 cgacggaaga ctctcctccg tgcgtcctcg tcttcaccgg tcgcgttcct gaaacgcaga
     3721 tgtgcctcgc gccgcactgc tccgaacaat aaagattcta caatactagc ttttatggtt
     3781 atgaagagga aaaattggca gtaacctggc cccacaaacc ttcaaattaa cgaatcaaat
     3841 taacaaccat aggatgataa tgcgattagt tttttagcct tatttctggg gtaattaatc
     3901 agcgaagcga tgatttttga tctattaaca gatatataaa tggaaaagct gcataaccac
     3961 tttaactaat actttcaaca ttttcagttt gtattacttc ttattcaaat gtcataaaag
     4021 tatcaacaaa aaattgttaa tatacctcta tactttaacg tcaaggagaa aaaaccccgg
     4081 attctagaac tagtggatcc cccatcacaa gtttgtacaa aaaagctgaa cgagaaacgt
     4141 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
     4201 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
     4261 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
     4321 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     4381 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     4441 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     4501 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
     4561 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     4621 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     4681 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     4741 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     4801 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     4861 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     4921 cgtctgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     4981 gtggcagggc ggggcgtaaa cgccgcgtgg atccggctta ctaaaagcca gataacagta
     5041 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
     5101 tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga cagcgacagc
     5161 tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc
     5221 agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg
     5281 ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac aggggctggt
     5341 gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat
     5401 gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca
     5461 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     5521 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     5581 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     5641 ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc atagtgactg
     5701 gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     5761 attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag tggtgatggg
     5821 ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagtcatg taattagtta
     5881 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     5941 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     6001 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     6061 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt
     6121 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     6181 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     6241 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     6301 cctgaatggc gaatggcgcg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6361 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     6421 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6481 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     6541 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6601 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     6661 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     6721 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcctg
     6781 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatagg caagtgcaca
     6841 aacaatactt aaataaatac tactcagtaa taacctattt cttagcattt ttgacgaaat
     6901 ttgctatttt gttagagtct tttacaccat ttgtctccac acctccgctt acatcaacac
     6961 caataacgcc atttaatcta agcgcatcac caacattttc tggcgtcagt ccaccagcta
     7021 acataaaatg taagctttcg gggctctctt gccttccaac ccagtcagaa atcgagttcc
     7081 aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa caagggaata aacgaatgag
     7141 gtttctgtga agctgcactg agtagtatgt tgcagtcttt tggaaatacg agtcttttaa
     7201 taactggcaa accgaggaac tcttggtatt cttgccacga ctcatctcca tgcagttgga
     7261 cgatatcaat gccgtaatca ttgaccagag ccaaaacatc ctccttaggt tgattacgaa
     7321 acacgccaac caagtatttc ggagtgcctg aactattttt atatgctttt acaagacttg
     7381 aaattttcct tgcaataacc gggtcaattg ttctctttct attgggcaca catataatac
     7441 ccagcaagtc agcatcggaa tctagagcac attctgcggc ctctgtgctc tgcaagccgc
     7501 aaactttcac caatggacca gaactacctg tgaaattaat aacagacata ctccaagctg
     7561 cctttgtgtg cttaatcacg tatactcacg tgctcaatag tcaccaatgc cctccctctt
     7621 ggccctctcc ttttcttttt tcgaccgaat taattcttaa tcggcaaaaa aagaaaagct
     7681 ccggatcaag attgtacgta aggtgacaag ctatttttca ataaagaata tcttccacta
     7741 ctgccatctg gcgtcataac tgcaaagtac acatatatta cgatgctgtc tattaaatgc
     7801 ttcctatatt atatatatag taatgtcgtt tatggtgcac tctcagtaca atctgctctg
     7861 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     7921 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     7981 gtcagaggtt ttcaccgtca tcaccgaaac gcgcga
//
