LOCUS       sequence-7947-ex        7673 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 7673)
  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 7673)
  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: pAG306GPD-ECFP-ccdB
FEATURES             Location/Qualifiers
     source          1..7673
                     /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"
     promoter        190..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; also known as the TDH3 promoter"
     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"
     CDS             join(2688..2690,2691..2693,2694..3404)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2733..2754)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2994..3013)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3341..3362
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3429..3553
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3578..3583,3584..3601,3602..3608)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3662..4321
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3728..3747)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4666..4971
                     /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     4850..4869
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5012..5136)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      5143..5390
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5159..5177)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5413..5431)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5452..5468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5452..5468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5465..5487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5476..5492
                     /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(5500..5506,5507..5524,5525..5530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5683..5700)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5854..6442)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6613..7404,7405..7473))
                     /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     7236..7255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7474..7578)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7646..7664
                     /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 ggatcccccg ggacaaaatg gtgagcaagg
     2701 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2761 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2821 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2881 tgacctgggg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     2941 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3001 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3061 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3121 actacatcag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     3181 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3241 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3301 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3361 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtgga ccaggtggtg
     3421 gacatcaaac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
     3481 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     3541 tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc
     3601 gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa
     3661 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
     3721 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
     3781 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
     3841 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
     3901 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     3961 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     4021 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     4081 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     4141 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     4201 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca
     4261 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     4321 aacgccgcgt ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga
     4381 tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat
     4441 gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca
     4501 tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc
     4561 tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta
     4621 ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt
     4681 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     4741 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     4801 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     4861 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     4921 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     4981 tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca
     5041 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     5101 ttacgtttct cgttcagctt tcttgtacaa agtggttcga tgtcatgtaa ttagttatgt
     5161 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     5221 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     5281 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     5341 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc
     5401 cagcttttgt tccctttagt gagggttaat tccgagcttg gcgtaatcat ggtcatagct
     5461 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacataggag ccggaagcat
     5521 aaagtgtaaa gcctggggtg cctaatgagt gaggtaactc acattaattg cgttgcgctc
     5581 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5641 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5701 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5761 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5821 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctcggcc cccctgacga
     5881 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5941 ccaggcgttc ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6001 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     6061 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6121 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6181 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6241 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     6301 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6361 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6421 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6481 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6541 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6601 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6661 tcgttcatcc atagttgcct gactgcccgt cgtgtagata actacgatac gggagggctt
     6721 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6781 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6841 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6901 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6961 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7021 gtgaaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7081 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7141 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7201 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7261 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7321 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7381 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7441 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7501 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7561 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7621 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
