LOCUS       sequence-7895-ex        6963 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 6963)
  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 6963)
  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: pAG304GPD-ccdB-HA
FEATURES             Location/Qualifiers
     source          1..6963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        185..465
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             466..1140
                     /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"
     rep_origin      complement(1237..1692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1374..1395)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1586..1605
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1822..1844
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1836..1853
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1837..1853
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1860..1879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1860..1878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1903..2547
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2441..2464
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     2553..2569
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2553..2569
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2577..2701
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2726..2731,2732..2749,2750..2756)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2810..3469
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2876..2895)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3814..4119
                     /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     3998..4017
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4160..4284)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4325..4414
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      4433..4680
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4449..4467)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4703..4721)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4742..4758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4742..4758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4755..4777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4766..4782
                     /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)."
     promoter        complement(join(4790..4796,4797..4814,4815..4820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4973..4990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5144..5732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5903..6694,6695..6763))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6526..6545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6764..6868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6936..6954
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accaaacgac attactatat atataatata ggaagcattt aatagacagc atcgtaatat
      241 atgtgtactt tgcagttatg acgccagatg gcagtagtgg aagatattct ttattgaaaa
      301 atagcttgtc accttacgta caatcttgat ccggagcttt tctttttttg ccgattaaga
      361 attaattcgg tcgaaaaaag aaaaggagag ggccaagagg gagggcattg gtgactattg
      421 agcacgtgag tatacgtgat taagcacaca aaggcagctt ggagtatgtc tgttattaat
      481 ttcacaggta gttctggtcc attggtgaaa gtttgcggct tgcagagcac agaggccgca
      541 gaatgtgctc tagattccga tgctgacttg ctgggtatta tatgtgtgcc caatagaaag
      601 agaacaattg acccggttat tgcaaggaaa atttcaagtc ttgtaaaagc atataaaaat
      661 agttcaggca ctccgaaata cttggttggc gtgtttcgta atcaacctaa ggaggatgtt
      721 ttggctctgg tcaatgatta cggcattgat atcgtccaac tgcatggaga tgagtcgtgg
      781 caagaatacc aagagttcct cggtttgcca gttattaaaa gactcgtatt tccaaaagac
      841 tgcaacatac tactcagtgc agcttcacag aaacctcatt cgtttattcc cttgtttgat
      901 tcagaagcag gtgggacagg tgaacttttg gattggaact cgatttctga ctgggttgga
      961 aggcaagaga gccccgaaag cttacatttt atgttagctg gtggactgac gccagaaaat
     1021 gttggtgatg cgcttagatt aaatggcgtt attggtgttg atgtaagcgg aggtgtggag
     1081 acaaatggtg taaaagactc taacaaaata gcaaatttcg tcaaaaatgc taagaaatag
     1141 gttattactg agtagtattt atttaagtat tgtttgtgca cttgcctgcg gtgtgaaata
     1201 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt
     1261 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1321 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1381 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     1441 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     1501 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     1561 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     1621 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     1681 tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     1741 tgcgggcctc ttcgctatta cgccagctgg cgaagggggg atgtgctgca aggcgattaa
     1801 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     1861 aatacgactc actatagggc gaattggagc tcttcgagtt tatcattatc aatactcgcc
     1921 atttcaaaga atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa
     1981 aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca
     2041 gaatatataa caccgaaggt gcctgggtga acaggtttat tcctggcatc cactaaatat
     2101 aatggagccc gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat
     2161 tgttttcttc accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca
     2221 ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag
     2281 taaatgatga cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc
     2341 tattaccttc tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc
     2401 ctgaaattat tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat
     2461 tctgtaaatc tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag
     2521 ttttaaaaca ccagaactta gtttcgacgg attctagaac tagtggatcc cccatcacaa
     2581 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
     2641 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
     2701 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
     2761 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     2821 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     2881 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     2941 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     3001 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     3061 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     3121 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     3181 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     3241 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     3301 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     3361 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat
     3421 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgccgcgtgg
     3481 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     3541 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     3601 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     3661 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     3721 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     3781 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     3841 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     3901 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     3961 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     4021 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     4081 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     4141 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     4201 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     4261 ttcagctttc ttgtacaaag tggtgatggg ctgcaggaat tcgatatcaa gcttaggtgg
     4321 aatgtaccca tacgatgttc ctgactatgc gggctatccc tatgacgtcc cggactatgc
     4381 aggatcctat ccatatgacg ttccagatta cgctgctcag tgctaactcg agtcatgtaa
     4441 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     4501 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     4561 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     4621 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     4681 ggccggtacc cagcttttgt tccctttagt gagggttaat tccgagcttg gcgtaatcat
     4741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacataggag
     4801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gaggtaactc acattaattg
     4861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctcggcc
     5161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5221 tataaagata ccaggcgttc ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat
     5341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5581 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5941 tctgtctatt tcgttcatcc atagttgcct gactgcccgt cgtgtagata actacgatac
     6001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6301 cccccatgtt gtgaaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     6901 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6961 gtc
//
