LOCUS       sequence-7994-ex        7474 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 7474)
  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 7474)
  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-EYFP-ccdB
FEATURES             Location/Qualifiers
     source          1..7474
                     /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"
     CDS             join(2489..2491,2492..2494,2495..3205)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2534..2555)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2795..2814)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3142..3163
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3230..3354
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3379..3384,3385..3402,3403..3409)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3463..4122
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3529..3548)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4467..4772
                     /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     4651..4670
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4813..4937)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4944..5191
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4960..4978)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(5214..5232)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5253..5269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5253..5269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5277..5293
                     /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(5301..5307,5308..5325,5326..5331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6414..7205,7206..7274))
                     /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     7037..7056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7275..7379)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7447..7465
                     /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 gggacaaaat ggtgagcaag ggcgaggagc tgttcaccgg
     2521 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2581 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2641 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg
     2701 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2761 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2821 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2881 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2941 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3001 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3061 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga
     3121 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3181 tctcggcatg gacgagctgt acaagggtgg accaggtggt ggacatcaaa caagtttgta
     3241 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     3301 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc
     3361 gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg
     3421 agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     3481 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     3541 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     3601 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     3661 atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat
     3721 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     3781 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     3841 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     3901 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     3961 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     4021 ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat
     4081 gaattacaac agtactgcga tgagtggcag ggcggggcgt aaacgccgcg tggatccggc
     4141 ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata
     4201 tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta
     4261 cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc
     4321 cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa
     4381 agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt
     4441 tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga
     4501 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga
     4561 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     4621 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     4681 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     4741 acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt
     4801 ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
     4861 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
     4921 ttcttgtaca aagtggttcg atgtcatgta attagttatg tcacgcttac attcacgccc
     4981 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     5041 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     5101 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     5161 ggttttggga cgctcgaagg ctttaatttg cggccggtac ccagcttttg ttccctttag
     5221 tgagggttaa ttccgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5281 tatccgctca caattccaca caacatagga gccggaagca taaagtgtaa agcctggggt
     5341 gcctaatgag tgaggtaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt cccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6481 tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa agcggttagc
     6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7381 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7441 tataaaaata ggcgtatcac gaggcccttt cgtc
//
