LOCUS       sequence-265036-        5598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRP1 based Yeast/E.coli shuttle vector designed to clone and express
            Cas9 programming spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5598)
  AUTHORS   Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, van den 
            Broek M, Daran-Lapujade P, Pronk JT, van Maris AJ, Daran JM
  TITLE     CRISPR/Cas9: a molecular Swiss army knife for simultaneous 
            introduction of multiple genetic modifications in Saccharomyces 
            cerevisiae.
  JOURNAL   FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi: 
            10.1093/femsyr/fov004. Epub 2015 Mar 4.
  PUBMED    25743786
REFERENCE   2  (bases 1 to 5598)
  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: "FEMS Yeast 
            Res."; date: "2015-03"; volume: "15(2). pii: fov004. doi"; pages: " 
            10.1093/femsyr/fov004. Epub 2015 Mar 4"
COMMENT     Sequence Label: pMEL17
FEATURES             Location/Qualifiers
     source          1..5598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        691..959
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        980..1054
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1059..1078
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      1084..1331
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1349..1368)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1350..1368)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1378..1395)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1378..1394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1387..1409)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1622..1641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(1913..2587)
                     /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(2588..2869)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(2890..2909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    3069..3116
                     /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)."
     rep_origin      3097..4079
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        4106..4210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4211..4279,4280..5071)
                     /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(4429..4448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5242..5598,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct tctttgaaaa gataatgtat gattatgctt
      721 tcactcatat ttatacagaa acttgatgtt ttctttcgag tatatacaag gtgattacat
      781 gtacgtttga agtacaactc tagattttgt agtgccctct tgggctagcg gtaaaggtgc
      841 gcattttttc acaccctaca atgttctgtt caaaagattt tggtcaaacg ctgtagaagt
      901 gaaagttggt gcgcatgttt cggcgttcga aacttctccg cagtgaaaga taaatgatcg
      961 atacgttctc tatggaggag ttttagagct agaaatagca agttaaaata aggctagtcc
     1021 gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt ttatgtcttc
     1081 gagtcatgta attagttatg tcacgcttac gttcacgccc tccccccaca tccgctctaa
     1141 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     1201 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     1261 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     1321 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     1381 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     1441 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1501 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     1561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1741 aaacttgatt agggtgatgg ttcacgtagt gggccgttga acattcttag gctggtcgaa
     1801 tcatttagac acgggcatcg tcctctcgaa aggtgcatct gtgcggtatt tcacaccgca
     1861 tagatcggca agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct
     1921 tagcattttt gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac
     1981 ctccgcttac atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg
     2041 gcgtcagtcc accagctaac ataaaatgta agctttcggg gctctcttgc cttccaaccc
     2101 agtcagaaat cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca
     2161 agggaataaa cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg
     2221 gaaatacgag tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact
     2281 catctccatg cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct
     2341 ccttaggttg attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat
     2401 atgcttttac aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat
     2461 tgggcacaca tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct
     2521 ctgtgctctg caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa
     2581 cagacatact ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc
     2641 accaatgccc tccctcttgg ccctctcctt ttcttttttc gaccgaatta attcttaatc
     2701 ggcaaaaaaa gaaaagctcc ggatcaagat tgtacgtaag gtgacaagct atttttcaat
     2761 aaagaatatc ttccactact gccatctggc gtcataactg caaagtacac atatattacg
     2821 atgctgttct attaaatgct tcctatatta tatatatagt aatgtcgttg atctatggtg
     2881 cactctcagt acaatctgct ctgatgccgc agtgcctatt gatgatctgg cggaatgtct
     2941 gccgtgccat agccatgcct tcacatatag tacaggcaac acgcagatat aggtgcgacg
     3001 tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct cgttttcgga
     3061 aacgctttga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcagag
     3121 cgcttttgaa aaccaaaagc gctctgaaga cgcactttca aaaaaccaaa aacgcaccgg
     3181 actgtaacga gctactaaaa tattgcgaat accgcttcca caaacattgc tcaaaagtat
     3241 ctctttgcta tatatctctg tgctatatcc ctatataacc tacccatcca cctttcgctc
     3301 cttgaacttg catctaaact cgacctctac attttttatg tttatctcta gtattactct
     3361 ttagacaaaa aaattgtagt aagaactatt catagagtga atcgaaaaca atacgaaaat
     3421 gtaaacattt cctatacgta gtatatagag acaaaataga agaaaccgtt cataattttc
     3481 tgaccaatga agaatcatca acgctatcac tttctgttca caaagtatgc gcaatccaca
     3541 tcggtataga atataatcgg ggatgccttt atcttgaaaa aatgcacccg cagcttcgct
     3601 agtaatcagt aaacgcggga agtggagtca ggcttttttt atggaagaga aaatagacac
     3661 caaagtagcc ttcttctaac cttaacggac ctacagtgca aaaagttatc aagagactgc
     3721 attatagagc gcacaaagga gaaaaaaagt aatctaagat gctttgttag aaaaatagcg
     3781 ctctcgggat gcatttttgt agaacaaaaa agaagtatag attctttgtt ggtaaaatag
     3841 cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg tttgaaaaat
     3901 tagcgctctc gcgttgcatt tttgttttac aaaaatgaag cacagattct tcgttggtaa
     3961 aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
     4021 aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat gaagcacaga tgcttcgttc
     4081 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4141 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4201 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4261 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4321 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4381 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4441 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4501 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4561 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4621 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4681 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4741 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4801 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4861 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4921 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4981 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5041 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5101 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5161 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5221 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5281 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5341 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5401 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5461 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5521 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5581 gcccagcttg gagcgaac
//
