LOCUS       sequence-7896-ex        8001 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8001)
  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 8001)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pAG305GAL-ccdB-HA
FEATURES             Location/Qualifiers
     source          1..8001
                     /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"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2474..2929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2823..2842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3059..3081
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3073..3090
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3074..3090
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3097..3116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3097..3115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3135..3576
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3135..3252
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3547..3570
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3591..3607
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3591..3607
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3615..3739
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3764..3769,3770..3787,3788..3794)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3848..4507
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3914..3933)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4852..5157
                     /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     5036..5055
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5198..5322)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5363..5452
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5471..5718
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5487..5505)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5741..5759)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5780..5796)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5780..5796)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5793..5815)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5804..5820
                     /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(5828..5834,5835..5852,5853..5858))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6011..6028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6182..6770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6941..7732,7733..7801))
                     /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     7564..7583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7802..7906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7974..7992
                     /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 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgagggtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
     2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3001 cagctggcga aggggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3061 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     3121 ttggagctct agtacggatt agaagccgcc gagcgggcga cagccctccg acggaagact
     3181 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
     3241 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
     3301 aattggcagt aacctggccc cacaaacctt caaattaacg aatcaaatta acaaccatag
     3361 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
     3421 atttttgatc tattaacaga tatataaatg gaaaagctgc ataaccactt taactaatac
     3481 tttcaacatt ttcagtttgt attacttctt attcaaatgt cataaaagta tcaacaaaaa
     3541 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat tctagaacta
     3601 gtggatcccc catcacaagt ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa
     3661 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     3721 aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc
     3781 cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga
     3841 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     3901 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     3961 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     4021 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
     4081 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     4141 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     4201 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     4261 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     4321 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     4381 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tctgtgatgg
     4441 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     4501 ggcgtaaacg ccgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     4561 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     4621 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     4681 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     4741 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     4801 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt
     4861 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     4921 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     4981 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     5041 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     5101 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     5161 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     5221 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     5281 atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgatgggct gcaggaattc
     5341 gatatcaagc ttaggtggaa tgtacccata cgatgttcct gactatgcgg gctatcccta
     5401 tgacgtcccg gactatgcag gatcctatcc atatgacgtt ccagattacg ctgctcagtg
     5461 ctaactcgag tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc
     5521 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
     5581 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac
     5641 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
     5701 tcgaaggctt taatttgcgg ccggtaccca gcttttgttc cctttagtga gggttaattc
     5761 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5821 ttccacacaa cataggagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5881 ggtaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5941 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6001 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6061 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6181 ttttccatag gctcggcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6241 ggcgaaaccc gacaggacta taaagatacc aggcgttccc ccctggaagc tccctcgtgc
     6301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6361 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6601 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     6661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctgcccgtcg
     7021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7321 caaggcgagt tacatgatcc cccatgttgt gaaaaaaagc ggttagctcc ttcggtcctc
     7381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     7981 gtatcacgag gccctttcgt c
//
