LOCUS       sequence-127808-        7574 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Yeast Gateway TRP1 2micron vector, no promoter (GPD1 promoter 
            removed).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7574)
  AUTHORS   Cherry PD, White LK, York K, Hesselberth JR
  TITLE     Genetic bypass of essential RNA repair enzymes in budding yeast.
  JOURNAL   RNA. 2018 Mar;24(3):313-323. doi: 10.1261/rna.061788.117. Epub 2017 
            Dec 6.
  PUBMED    29212664
REFERENCE   2  (bases 1 to 7574)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1261/rna.061788.117"; journalName: "RNA"; date: "2018-03"; 
            volume: "24"; issue: "3"; pages: "313-323"
COMMENT     Sequence Label: pAG424-ccdB
FEATURES             Location/Qualifiers
     source          1..7574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..36
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     20..36
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    44..168
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(193..198,199..216,217..223)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             277..936
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(343..362)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1281..1586
                     /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     1465..1484
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1627..1751)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1784..1800)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1785..1801)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1803..2050
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1819..1837)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2068..2087)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2069..2087)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2097..2114)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2097..2113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2106..2128)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2258..2713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2345..2364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2555..2576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2812..3486)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(3487..3767)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3784..3803)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3903..3925
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3963..3981)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4022..5364
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4354..4401
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        5391..5495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5496..5564,5565..6356)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5714..5733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6527..7115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7016..7035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7269..7286
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(7439..7444,7445..7462,7463..7469)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7477..7493
                     /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)."
     primer_bind     7482..7504
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7501..7517
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7501..7517
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7536..7556
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7538..7556
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gaacttagtt tcgacggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa
       61 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa
      121 aaaacagact acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta
      181 ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag
      241 gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
      301 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
      361 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
      421 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
      481 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
      541 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
      601 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
      661 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
      721 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
      781 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
      841 attcaggttc atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct taatgaatta
      901 caacagtact gcgatgagtg gcagggcggg gcgtaaacgc cgcgtggatc cggcttacta
      961 aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact
     1021 gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga
     1081 cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct
     1141 ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga
     1201 aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga
     1261 cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt
     1321 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga
     1381 tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg
     1441 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     1501 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     1561 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag
     1621 gtcgaccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     1681 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg
     1741 tacaaagtgg tgatgggctg caggaattcg atatcaagct tatcgatacc gtcgacctcg
     1801 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     1861 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     1921 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     1981 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     2041 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2101 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2161 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2221 tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta
     2281 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2341 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2401 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2461 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2521 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2581 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2641 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2701 acgtttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2761 gcataggcaa gtgcacaaac aatacttaaa taaatactac tcagtaataa cctatttctt
     2821 agcatttttg acgaaatttg ctattttgtt agagtctttt acaccatttg tctccacacc
     2881 tccgcttaca tcaacaccaa taacgccatt taatctaagc gcatcaccaa cattttctgg
     2941 cgtcagtcca ccagctaaca taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca
     3001 gtcagaaatc gagttccaat ccaaaagttc acctgtccca cctgcttctg aatcaaacaa
     3061 gggaataaac gaatgaggtt tctgtgaagc tgcactgagt agtatgttgc agtcttttgg
     3121 aaatacgagt cttttaataa ctggcaaacc gaggaactct tggtattctt gccacgactc
     3181 atctccatgc agttggacga tatcaatgcc gtaatcattg accagagcca aaacatcctc
     3241 cttaggttga ttacgaaaca cgccaaccaa gtatttcgga gtgcctgaac tatttttata
     3301 tgcttttaca agacttgaaa ttttccttgc aataaccggg tcaattgttc tctttctatt
     3361 gggcacacat ataataccca gcaagtcagc atcggaatct agagcacatt ctgcggcctc
     3421 tgtgctctgc aagccgcaaa ctttcaccaa tggaccagaa ctacctgtga aattaataac
     3481 agacatactc caagctgcct ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca
     3541 ccaatgccct ccctcttggc cctctccttt tcttttttcg accgaattaa ttcttaatcg
     3601 gcaaaaaaag aaaagctccg gatcaagatt gtacgtaagg tgacaagcta tttttcaata
     3661 aagaatatct tccactactg ccatctggcg tcataactgc aaagtacaca tatattacga
     3721 tgctgtctat taaatgcttc ctatattata tatatagtaa tgtcgtttat ggtgcactct
     3781 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     3841 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3901 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     3961 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttaata
     4021 tgatccaata tcaaaggaaa tgatagcatt gaaggatgag actaatccaa ttgaggagtg
     4081 gcagcatata gaacagctaa agggtagtgc tgaaggaagc atacgatacc ccgcatggaa
     4141 tgggataata tcacaggagg tactagacta cctttcatcc tacataaata gacgcatata
     4201 agtacgcatt taagcataaa cacgcactat gccgttcttc tcatgtatat atatatacag
     4261 gcaacacgca gatataggtg cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc
     4321 attttcggaa gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa gttcctattc
     4381 tctagaaagt ataggaactt cagagcgctt ttgaaaacca aaagcgctct gaagacgcac
     4441 tttcaaaaaa ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc
     4501 ttccacaaac attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata
     4561 taacctaccc atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt
     4621 ttatgtttat ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag
     4681 agtgaatcga aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa
     4741 atagaagaaa ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct
     4801 gttcacaaag tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt
     4861 gaaaaaatgc acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt
     4921 tttttatgga agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca
     4981 gtgcaaaaag ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct
     5041 aagatgcttt gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag
     5101 tatagattct ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc
     5161 agctcagatt ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa
     5221 tgaagcacag attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa
     5281 aatgcagctc agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac
     5341 aaaatgaagc acagatgctt cgttcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5401 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5461 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5521 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5581 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5641 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5701 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5761 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5821 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5881 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5941 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6001 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6061 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6121 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6181 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6241 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6301 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6361 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6421 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6481 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6541 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6601 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6661 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6721 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6781 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6841 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6901 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6961 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7021 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7081 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7141 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7201 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7261 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7321 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7381 cgggcagtga gcgcaacgca attaatgtga gttacctcac tcattaggca ccccaggctt
     7441 tacactttat gcttccggct cctatgttgt gtggaattgt gagcggataa caatttcaca
     7501 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     7561 aaaagctgga gctc
//
