LOCUS       sequence-128474-        7668 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 7668)
  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 7668)
  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: pAG413GPD-ccdB-HA
FEATURES             Location/Qualifiers
     source          1..7668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /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(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2804..3447
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3341..3364
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3453..3469
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3453..3469
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3477..3601
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3626..3631,3632..3649,3650..3656)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3710..4369
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3776..3795)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4714..5019
                     /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     4898..4917
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5060..5184)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5225..5314
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5333..5580
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5349..5367)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5598..5617)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5599..5617)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5627..5644)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5627..5643)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5636..5658)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5788..6243
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5875..5894)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6085..6106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6503..7165)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(7166..7352)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(7499..7518)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7618..7640
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctcttcgagt ttatcattat caatactcgc
     2821 catttcaaag aatacgtaaa taattaatag tagtgatttt cctaacttta tttagtcaaa
     2881 aaattagcct tttaattctg ctgtaacccg tacatgccca aaataggggg cgggttacac
     2941 agaatatata acatcgtagg tgtctgggtg aacagtttat tcctggcatc cactaaatat
     3001 aatggagccc gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat
     3061 tgttttcttc accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca
     3121 ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag
     3181 taaatgatga cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc
     3241 tattaccttc tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc
     3301 ctgaaattat tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat
     3361 tctgtaaatc tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag
     3421 ttttaaaaca ccagaactta gtttcgacgg attctagaac tagtggatcc cccatcacaa
     3481 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
     3541 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
     3601 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
     3661 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     3721 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     3781 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     3841 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     3901 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     3961 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     4021 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     4081 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     4141 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     4201 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     4261 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat
     4321 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgccgcgtgg
     4381 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     4441 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     4501 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     4561 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     4621 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     4681 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     4741 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     4801 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     4861 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     4921 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     4981 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     5041 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     5101 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     5161 ttcagctttc ttgtacaaag tggtgatggg ctgcaggaat tcgatatcaa gcttaggtgg
     5221 aatgtaccca tacgatgttc ctgactatgc gggctatccc tatgacgtcc cggactatgc
     5281 aggatcctat ccatatgacg ttccagatta cgctgctcag tgctaactcg agtcatgtaa
     5341 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     5401 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     5461 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     5521 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     5581 ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     5641 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5701 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5761 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg
     5821 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5881 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5941 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     6001 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     6061 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     6121 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6181 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     6241 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagatcc
     6301 gtcgagttca agagaaaaaa aaagaaaaag caaaaagaaa aaaggaaagc gcgcctcgtt
     6361 cagaatgaca cgtatagaat gatgcattac cttgtcatct tcagtatcat actgttcgta
     6421 tacatactta ctgacattca taggtataca tatatacaca tgtatatata tcgtatgctg
     6481 cagctttaaa taatcggtgt cactacataa gaacaccttt ggtggaggga acatcgttgg
     6541 taccattggg cgaggtggct tctcttatgg caaccgcaag agccttgaac gcactctcac
     6601 tacggtgatg atcattcttg cctcgcagac aatcaacgtg gagggtaatt ctgctagcct
     6661 ctgcaaagct ttcaagaaaa tgcgggatca tctcgcaaga gagatctcct actttctccc
     6721 tttgcaaacc aagttcgaca actgcgtacg gcctgttcga aagatctacc accgctctgg
     6781 aaagtgcctc atccaaaggc gcaaatcctg atccaaacct ttttactcca cgcacggccc
     6841 ctagggcctc tttaaaagct tgaccgagag caatcccgca gtcttcagtg gtgtgatggt
     6901 cgtctatgtg taagtcacca atgcactcaa cgattagcga ccagccggaa tgcttggcca
     6961 gagcatgtat catatggtcc agaaacccta tacctgtgtg gacgttaatc acttgcgatt
     7021 gtgtggcctg ttctgctact gcttctgcct ctttttctgg gaagatcgag tgctctatcg
     7081 ctaggggacc accctttaaa gagatcgcaa tctgaatctt ggtttcattt gtaatacgct
     7141 ttactagggc tttctgctct gtcatctttg ccttcgttta tcttgcctgc tcatttttta
     7201 gtatattctt cgaagaaatc acattacttt atataatgta taattcatta tgtgataatg
     7261 ccaatcgcta agaaaaaaaa agagtcatcc gctaggggaa aaaaaaaaat gaaaatcatt
     7321 accgaggcat aaaaaaatat agagtgtact agaggaggcc aagagtaata gaaaaagaaa
     7381 attgcgggaa aggactgtgt tatgacttcc ctgactaatg ccgtgttcaa acgatacctg
     7441 gcagtgactc ctagcgctca ccaagctctt aaaacgggaa tttatggtgc actctcagta
     7501 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cgcgctgacg
     7561 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     7621 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
