LOCUS       Exported                8200 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAG305GPD-ccdB-HA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8200)
  TITLE     S cerevisiae Advanced Gateway Destination Vectors
REFERENCE   2  (bases 1 to 8200)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8200
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        3140..3784
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     3678..3701
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3790..3806
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3790..3806
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3814..3938
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3963..3993
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4047..4706
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4113..4132)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5051..5356
                     /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     5235..5254
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5397..5521)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5562..5651
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5670..5917
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5686..5704)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5940..5958)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5979..5995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5979..5995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5992..6014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6003..6019
                     /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(6027..6057)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6210..6227)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6381..6969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7140..8000)
                     /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     7763..7782
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8001..8105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8173..8191
                     /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 tcgagtttat cattatcaat actcgccatt tcaaagaata cgtaaataat
     3181 taatagtagt gattttccta actttattta gtcaaaaaat tagcctttta attctgctgt
     3241 aacccgtaca tgcccaaaat agggggcggg ttacacagaa tatataacac cgaaggtgcc
     3301 tgggtgaaca ggtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg
     3361 gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt
     3421 tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg
     3481 ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg
     3541 acccacgcat gtatctatct cattttctta caccttctat taccttctgc tctctctgat
     3601 ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac
     3661 taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact
     3721 tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca gaacttagtt
     3781 tcgacggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa agctgaacga
     3841 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
     3901 acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag
     3961 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga
     4021 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     4081 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     4141 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
     4201 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     4261 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     4321 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     4381 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     4441 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     4501 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     4561 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     4621 atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     4681 gcgatgagtg gcagggcggg gcgtaaacgc cgcgtggatc cggcttacta aaagccagat
     4741 aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata
     4801 cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag
     4861 cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca
     4921 accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa
     4981 gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg
     5041 ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     5101 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     5161 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     5221 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     5281 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     5341 gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     5401 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     5461 ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg
     5521 tgatgggctg caggaattcg atatcaagct taggtggaat gtacccatac gatgttcctg
     5581 actatgcggg ctatccctat gacgtcccgg actatgcagg atcctatcca tatgacgttc
     5641 cagattacgc tgctcagtgc taactcgagt catgtaatta gttatgtcac gcttacattc
     5701 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     5761 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     5821 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     5881 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccag cttttgttcc
     5941 ctttagtgag ggttaattcc gagcttggcg taatcatggt catagctgtt tcctgtgtga
     6001 aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa gtgtaaagcc
     6061 tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6121 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6181 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6241 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6301 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6361 aaggccgcgt tgctggcgtt tttccatagg ctcggccccc ctgacgagca tcacaaaaat
     6421 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgttcccc
     6481 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6541 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     6601 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6661 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6721 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6781 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6841 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6901 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6961 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7021 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7081 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7141 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7201 gttgcctgac tgcccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7261 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7321 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7381 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7441 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7501 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg aaaaaaagcg
     7561 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7621 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7681 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7741 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7801 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7861 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7921 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7981 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     8041 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     8101 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     8161 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
