LOCUS       Exported                7678 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAG306GPD-ccdB-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7678)
  TITLE     S cerevisiae Advanced Gateway Destination Vectors
REFERENCE   2  (bases 1 to 7678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7678
                     /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..5141
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             4422..5138
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     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      5148..5395
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5164..5182)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5418..5436)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5457..5473)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5457..5473)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5470..5492)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5481..5497
                     /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(5505..5535)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5688..5705)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5859..6447)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6618..7478)
                     /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     7241..7260
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7479..7583)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7651..7669
                     /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 acggcgtgca 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 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     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 tgtacaagta actcgagtca tgtaattagt
     5161 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     5221 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     5281 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     5341 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg
     5401 gtacccagct tttgttccct ttagtgaggg ttaattccga gcttggcgta atcatggtca
     5461 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat aggagccgga
     5521 agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt aactcacatt aattgcgttg
     5581 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5641 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5701 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5761 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5821 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct cggcccccct
     5881 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5941 agataccagg cgttcccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6001 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     6061 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6121 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6181 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6241 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6301 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6361 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6421 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6481 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6541 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6601 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6661 ctatttcgtt catccatagt tgcctgactg cccgtcgtgt agataactac gatacgggag
     6721 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6781 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6841 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6901 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6961 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7021 atgttgtgaa aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7081 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7141 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7201 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7261 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7321 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7381 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7441 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7501 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7561 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     7621 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
