LOCUS       sequence-8042-ex        7479 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 7479)
  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 7479)
  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: pAG306GAL-ccdB-Cerulean
FEATURES             Location/Qualifiers
     source          1..7479
                     /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        2004..2445
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2004..2121
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2416..2439
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     2460..2476
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2460..2476
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2484..2608
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2633..2638,2639..2656,2657..2663)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2717..3376
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2783..2802)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3721..4026
                     /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     3905..3924
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4067..4191)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(4223..4225,4226..4228,4229..4939)
                     /codon_start=1
                     /product="monomeric optimized variant of ECFP (Kremers et 
                     al., 2006)"
                     /label=SCFP3A
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4268..4289)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4529..4548)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4876..4897
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      4949..5196
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4965..4983)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5219..5237)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5258..5274)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5258..5274)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5271..5293)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5282..5298
                     /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(5306..5312,5313..5330,5331..5336))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5489..5506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5660..6248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6419..7210,7211..7279))
                     /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     7042..7061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7280..7384)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7452..7470
                     /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 tggagctcta gtacggatta gaagccgccg agcgggcgac agccctccga
     2041 cggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt
     2101 gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg
     2161 aagaggaaaa attggcagta acctggcccc acaaaccttc aaattaacga atcaaattaa
     2221 caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc
     2281 gaagcgatga tttttgatct attaacagat atataaatgg aaaagctgca taaccacttt
     2341 aactaatact ttcaacattt tcagtttgta ttacttctta ttcaaatgtc ataaaagtat
     2401 caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatt
     2461 ctagaactag tggatccccc atcacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
     2521 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     2581 gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact
     2641 ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg
     2701 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     2761 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     2821 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     2881 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
     2941 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     3001 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     3061 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     3121 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     3181 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     3241 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     3301 ctgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     3361 gcagggcggg gcgtaaacgc cgcgtggatc cggcttacta aaagccagat aacagtatgc
     3421 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
     3481 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
     3541 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
     3601 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
     3661 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
     3721 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     3781 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     3841 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     3901 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
     3961 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     4021 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat
     4081 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     4141 gatatttata tcattttacg tttctcgttc agctttcttg tacaaagtgg tgatgggctg
     4201 caggaattcg atatcaagct taatggtgag caagggcgag gagctgttca ccggggtggt
     4261 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     4321 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4381 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcgc
     4441 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     4501 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     4561 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     4621 ggacggcaac atcctggggc acaagctgga gtacaactac atcagcgaca acgtctatat
     4681 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     4741 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     4801 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     4861 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     4921 catggacgag ctgtacaagt aactcgagtc atgtaattag ttatgtcacg cttacattca
     4981 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     5041 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     5101 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     5161 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccagc ttttgttccc
     5221 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5281 attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag tgtaaagcct
     5341 ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc
     5401 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5461 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5641 aggccgcgtt gctggcgttt ttccataggc tcggcccccc tgacgagcat cacaaaaatc
     5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgttccccc
     5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5821 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6061 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6481 ttgcctgact gcccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtga aaaaaagcgg
     6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7381 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7441 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
