LOCUS       sequence-208546-        5465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HIS3 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 5465)
  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 5465)
  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: pROS16
FEATURES             Location/Qualifiers
     source          1..5465
                     /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     2317..2336
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2483..2669
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2670..3332
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        3657..3925
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3946..4020
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4025..4044
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4269..5250
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5296..5315)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5320..5394)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5415..5465,1..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 gcactgcggc atcagagcag attgtactga
     2341 gagtgcacca taaattcccg ttttaagagc ttggtgagcg ctaggagtca ctgccaggta
     2401 tcgtttgaac acggcattag tcagggaagt cataacacag tcctttcccg caattttctt
     2461 tttctattac tcttggcctc ctctagtaca ctctatattt ttttatgcct cggtaatgat
     2521 tttcattttt tttttccacc tagcggatga ctcttttttt ttcttagcga ttggcattat
     2581 cacataatga attatacatt atataaagta atgtgatttc ttcgaagaat atactaaaaa
     2641 atgagcaggc aagataaacg aaggcaaaga tgacagagca gaaagcccta gtaaagcgta
     2701 ttacaaatga aaccaagatt cagattgcga tctctttaaa gggtggtccc ctagcgatag
     2761 agcactcgat cttcccagaa aaagaggcag aagcagtagc agaacaggcc acacaatcgc
     2821 aagtgattaa cgtccacaca ggtatagggt ttctggacca tatgatacat gctctggcca
     2881 agcattccgg ctggtcgcta atcgttgagt gcattggtga cttacacata gacgaccatc
     2941 acaccactga agactgcggg attgctctcg gtcaagcttt taaagaggcc ctaggggccg
     3001 tgcgtggagt aaaaaggttt ggatcaggat ttgcgccttt ggatgaggca ctttccagag
     3061 cggtggtaga tctttcgaac aggccgtacg cagttgtcga acttggtttg caaagggaga
     3121 aagtaggaga tctctcttgc gagatgatcc cgcattttct tgaaagcttt gcagaggcta
     3181 gcagaattac cctccacgtt gattgtctgc gaggcaagaa tgatcatcac cgtagtgaga
     3241 gtgcgttcaa ggctcttgcg gttgccataa gagaagccac ctcgcccaat ggtaccaacg
     3301 atgttccctc caccaaaggt gttcttatgt agtgacaccg attatttaaa gctgcagcat
     3361 acgatatata tacatgtgta tatatgtata cctatgaatg tcagtaagta tgtatacgaa
     3421 cagtatgata ctgaagatga caaggtaatg catcattcta tacgtgtcat tctgaacgag
     3481 gcgcgctttc cttttttctt tttgcttttt cttttttttt ctcttgaact cgacggatct
     3541 atgcggtgtg aaataccgca cagatgcacc tttcgagagg acgatgcccg tgtctaaatg
     3601 attcgaccag cctaagaatg ttcaacccct cactaaaggg aacaaaagct ggagcttctt
     3661 tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt gatgttttct
     3721 ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga ttttgtagtg
     3781 ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt tctgttcaaa
     3841 agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact
     3901 tctccgcagt gaaagataaa tgatcacttg aagattcttt agtgtgtttt agagctagaa
     3961 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     4021 gtgctttttt tgttttttat gtcttcgagt catgtaatta gttatgtcac gcttacgttc
     4081 acgccctcca cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat
     4141 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg
     4201 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc
     4261 ctattctgta gaaagtatag gaacctcaga gcgcttttga aaaccaaaag cgctctgaag
     4321 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
     4381 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
     4441 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
     4501 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
     4561 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
     4621 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
     4681 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
     4741 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
     4801 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
     4861 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
     4921 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
     4981 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
     5041 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
     5101 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
     5161 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
     5221 ttctacaaaa tgaagcacag atgcttcgtt ggagggcgtg aacgtaagcg tgacataact
     5281 aattacatga ctcgaagaca taaaaaacaa aaaaagcacc accgactcgg tgccactttt
     5341 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaactcctcc
     5401 atagagaacg tatcgatcat ttatctttca ctgcggagaa gtttcgaacg ccgaaacatg
     5461 cgcac
//
