LOCUS       Exported                7687 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAG306GPD-ccdB-ECFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7687)
  TITLE     S cerevisiae Advanced Gateway Destination Vectors
REFERENCE   2  (bases 1 to 7687)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7687
                     /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"
     promoter        195..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             411..1214
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1343..1798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1480..1501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1692..1711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1928..1950
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1942..1959
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1943..1959
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2009..2653
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     2547..2570
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     2659..2675
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2659..2675
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2683..2807
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2832..2862
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2916..3575
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2982..3001)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3920..4225
                     /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     4104..4123
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4266..4390)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4422..5138
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4467..4488)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4728..4747)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5075..5096
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      5157..5404
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5173..5191)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5427..5445)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5466..5482)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5466..5482)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5479..5501)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5490..5506
                     /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(5514..5544)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5697..5714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5868..6456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6627..7487)
                     /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     7250..7269
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7488..7592)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7660..7678
                     /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 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt
     1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta
     1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca
     1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     1981 tagggcgaat tggagctctt cgagtttatc attatcaata ctcgccattt caaagaatac
     2041 gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt agccttttaa
     2101 ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat atataacacc
     2161 gaaggtgcct gggtgaacag gtttattcct ggcatccact aaatataatg gagcccgctt
     2221 tttaagctgg catccagaaa aaaaaagaat cccagcacca aaatattgtt ttcttcacca
     2281 accatcagtt cataggtcca ttctcttagc gcaactacag agaacagggg cacaaacagg
     2341 caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa tgatgacaca
     2401 aggcaattga cccacgcatg tatctatctc attttcttac accttctatt accttctgct
     2461 ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga aattattccc
     2521 ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg taaatctatt
     2581 tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt aaaacaccag
     2641 aacttagttt cgacggattc tagaactagt ggatccccca tcacaagttt gtacaaaaaa
     2701 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     2761 aaacagacta cataatactg taaaacacaa catatccagt cactatggcg gccgcattag
     2821 gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg
     2881 atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     2941 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     3001 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     3061 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     3121 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     3181 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     3241 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     3301 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     3361 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     3421 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     3481 ttcaggttca tcatgccgtc tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     3541 aacagtactg cgatgagtgg cagggcgggg cgtaaacgcc gcgtggatcc ggcttactaa
     3601 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     3661 atatgtatac ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac
     3721 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     3781 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa
     3841 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     3901 gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     3961 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     4021 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     4081 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     4141 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     4201 taacctgatg ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg
     4261 tcgaccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     4321 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt
     4381 acaaagtggt gatgggctgc aggaattcga tatcaagctt aatggtgagc aagggcgagg
     4441 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4501 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4561 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4621 ggggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4681 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4741 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4801 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4861 tcagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca
     4921 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4981 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5041 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5101 ccgccgggat cactctcggc atggacgagc tgtacaagct gtacaagtaa ctcgagtcat
     5161 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa
     5221 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     5281 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     5341 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat
     5401 ttgcggccgg tacccagctt ttgttccctt tagtgagggt taattccgag cttggcgtaa
     5461 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5521 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta
     5581 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5641 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5701 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5761 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5821 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5881 ggcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5941 ggactataaa gataccaggc gttcccccct ggaagctccc tcgtgcgctc tcctgttccg
     6001 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6061 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6121 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6181 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6241 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6301 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6361 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6421 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6481 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6541 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6601 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6661 gcgatctgtc tatttcgttc atccatagtt gcctgactgc ccgtcgtgta gataactacg
     6721 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6781 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6841 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6901 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6961 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7021 tgatccccca tgttgtgaaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7081 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7141 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7201 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7261 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7321 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7381 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7441 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7501 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7561 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7621 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7681 tttcgtc
//
