LOCUS       sequence-208549-        5288 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 5288)
  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 5288)
  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: pROS17
FEATURES             Location/Qualifiers
     source          1..5288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(351..368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(522..1110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(602..621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1281..2072,2073..2141))
                     /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     1904..1923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2142..2246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2316..2335
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2356..2637
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             2638..3312
                     /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        3480..3748
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3769..3843
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3848..3867
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4092..5073
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5119..5138)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5143..5217)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5238..5288,1..218))
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
ORIGIN
        1 caactttcac ttctacagcg tttgaccaaa atcttttgaa cagaacattg tagggtgtga
       61 aaaaatgcgc acctttaccg ctagcccaag agggcactac aaaatctaga gttgtacttc
      121 aaacgtacat gtaatcacct tgtatatact cgaaagaaaa catcaagttt ctgtataaat
      181 atgagtgaaa gcataatcat acattatctt ttcaaagaag ctccagcttt tgttcccttt
      241 agtgagggta ttcacgtaga cggataggta tagccagaca tcagcagcat acttcgggaa
      301 ccgtaggctc tagagagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
      361 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      421 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      481 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      541 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      601 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      661 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      721 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      781 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      841 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      901 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      961 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1021 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1081 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1141 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1201 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1261 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1321 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1381 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1441 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1501 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1561 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1621 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1681 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1741 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1801 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1861 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1921 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1981 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2041 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2101 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2161 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2221 aaaaataaac aaataggggt tccgcgcact cgagactata tgtgaaggca tggctatggc
     2281 acggcagaca ttccgccaga tcatcaatag gcacgcggca tcagagcaga ttgtactgag
     2341 agtgcaccat agatcaacga cattactata tatataatat aggaagcatt taatagaaca
     2401 gcatcgtaat atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt
     2461 ctttattgaa aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt
     2521 tgccgattaa gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat
     2581 tggtgactat tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg
     2641 tctgttatta atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc
     2701 acagaggccg cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg
     2761 cccaatagaa agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa
     2821 gcatataaaa atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct
     2881 aaggaggatg ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga
     2941 gatgagtcgt ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta
     3001 tttccaaaag actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt
     3061 cccttgtttg attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct
     3121 gactgggttg gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg
     3181 acgccagaaa atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc
     3241 ggaggtgtgg agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat
     3301 gctaagaaat aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccga
     3361 tctatgcggt gtgaaatacc gcacagatgc acctttcgag aggacgatgc ccgtgtctaa
     3421 atgattcgac cagcctaaga atgttcaacc cctcactaaa gggaacaaaa gctggagctt
     3481 ctttgaaaag ataatgtatg attatgcttt cactcatatt tatacagaaa cttgatgttt
     3541 tctttcgagt atatacaagg tgattacatg tacgtttgaa gtacaactct agattttgta
     3601 gtgccctctt gggctagcgg taaaggtgcg cattttttca caccctacaa tgttctgttc
     3661 aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg cgcatgtttc ggcgttcgaa
     3721 acttctccgc agtgaaagat aaatgatcac ttgaagattc tttagtgtgt tttagagcta
     3781 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3841 gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa ttagttatgt cacgcttacg
     3901 ttcacgccct ccacgcattt aagcataaac acgcactatg ccgttcttct catgtatata
     3961 tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
     4021 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
     4081 ttcctattct gtagaaagta taggaacctc agagcgcttt tgaaaaccaa aagcgctctg
     4141 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
     4201 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
     4261 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
     4321 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
     4381 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
     4441 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
     4501 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
     4561 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
     4621 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
     4681 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
     4741 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
     4801 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
     4861 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     4921 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
     4981 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     5041 ttgttctaca aaatgaagca cagatgcttc gttggagggc gtgaacgtaa gcgtgacata
     5101 actaattaca tgactcgaag acataaaaaa caaaaaaagc accaccgact cggtgccact
     5161 ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct ctaaaactcc
     5221 tccatagaga acgtatcgat catttatctt tcactgcgga gaagtttcga acgccgaaac
     5281 atgcgcac
//
