LOCUS       sequence-214969-        5462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Cas9 programming sgRNA2 and sgRNA1 targetting GAL2 and
            HXT4-1-5/ HXT3-6-7 respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5462)
  AUTHORS   Wijsman M, Swiat MA, Marques WL, Hettinga JK, van den Broek M, Torre
            Cortes P, Mans R, Pronk JT, Daran JM, Daran-Lapujade P
  TITLE     A toolkit for rapid CRISPR-SpCas9 assisted construction of 
            hexose-transport-deficient Saccharomyces cerevisiae strains.
  JOURNAL   FEMS Yeast Res. 2019 Jan 1;19(1). pii: 5114578. doi: 
            10.1093/femsyr/foy107.
  PUBMED    30285096
REFERENCE   2  (bases 1 to 5462)
  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: "2019-01-1"; volume: "19(1). pii: 5114578. doi"; pages:
            " 10.1093/femsyr/foy107"
COMMENT     Sequence Label: pUDR295
FEATURES             Location/Qualifiers
     source          1..5462
                     /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..3329
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        3654..3922
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3943..4017
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4022..4041
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4087..5068
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5050..5097)
                     /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)."
     terminator      complement(5293..5312)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5317..5391)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5412..5462,1..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 ttttttcccc 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 ctactggcgc
     3001 gtggagtaaa aaggtttgga tcaggatttg cgcctttgga tgaggcactt tccagagcgg
     3061 tggtagatct ttcgaacagg ccgtacgcag ttgtcgaact tggtttgcaa agggagaaag
     3121 taggagatct ctcttgcgag atgatcccgc attttcttga aagctttgca gaggctagca
     3181 gaattaccct ccacgttgat tgtctgcgag gcaagaatga tcatcaccgt agtgagagtg
     3241 cgttcaaggc tcttgcggtt gccataagag aagccacctc gcccaatggt accaacgatg
     3301 ttccctccac caaaggtgtt cttatgtagt gacaccgatt atttaaagct gcagcatacg
     3361 atatatatac atgtgtatat atgtatacct atgaatgtca gtaagtatgt atacgaacag
     3421 tatgatactg aagatgacaa ggtaatgcat cattctatac gtgtcattct gaacgaggcg
     3481 cgctttcctt ttttcttttt gctttttctt tttttttctc ttgaactcga cggatctatg
     3541 cggtgtgaaa taccgcacag atgcaccttt cgagaggacg atgcccgtgt ctaaatgatt
     3601 cgaccagcct aagaatgttc aacccctcac taaagggaac aaaagctgga gcttctttga
     3661 aaagataatg tatgattatg ctttcactca tatttataca gaaacttgat gttttctttc
     3721 gagtatatac aaggtgatta catgtacgtt tgaagtacaa ctctagattt tgtagtgccc
     3781 tcttgggcta gcggtaaagg tgcgcatttt ttcacaccct acaatgttct gttcaaaaga
     3841 ttttggtcaa acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct
     3901 ccgcagtgaa agataaatga tcttatttat gtgaggtgaa tagttttaga gctagaaata
     3961 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtggtg
     4021 ctttttttgt tttttatgtc ttcgagtcat gtaattagtt atgtcacgct tacgttcacg
     4081 ccctccaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta
     4141 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg
     4201 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag
     4261 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
     4321 gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc
     4381 gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct
     4441 ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta
     4501 ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga
     4561 ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat tatattctat
     4621 accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt
     4681 ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg
     4741 tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg
     4801 tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt
     4861 tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca
     4921 aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt
     4981 acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa
     5041 aagcgctctg aagttcctat actttctaca gaataggaac ttcggaatag gaacttcaaa
     5101 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact
     5161 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc
     5221 atagtgcgtg tttatgctta aatgcgtgga gggcgtgaac gtaagcgtga cataactaat
     5281 tacatgactc gaagacataa aaaacaaaaa aagcaccacc gactcggtgc cactttttca
     5341 agttgataac ggactagcct tattttaact tgctatttct agctctaaaa ctagtagaag
     5401 aaatagttat cgatcattta tctttcactg cggagaagtt tcgaacgccg aaacatgcgc
     5461 ac
//
