LOCUS       sequence-220905-        5575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Cas9 programming sgRNA5 and sgRNA2 targetting 
            HXT13-15-16 and HXT2 respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5575)
  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 5575)
  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: pURD214
FEATURES             Location/Qualifiers
     source          1..5575
                     /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
     CDS             complement(2453..3256)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3257..3477)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3503..3522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3622..3644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3769..4037
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        4058..4132
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4137..4156
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4202..5183
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5406..5425)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5430..5504)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5525..5575,1..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 gcacgcggcc gccctgatgc ggtattttct
     2341 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     2401 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     2461 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     2521 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     2581 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     2641 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     2701 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     2761 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     2821 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     2881 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     2941 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     3001 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     3061 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     3121 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     3181 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     3241 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     3301 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     3361 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     3421 ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aattgaaaag
     3481 ctgtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3541 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3601 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3661 cgaaacgcgc gacagctgca cctttcgaga ggacgatgcc cgtgtctaaa tgattcgacc
     3721 agcctaagaa tgttcaaccc ctcactaaag ggaacaaaag ctggagcttc tttgaaaaga
     3781 taatgtatga ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta
     3841 tatacaaggt gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg
     3901 ggctagcggt aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg
     3961 gtcaaacgct gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca
     4021 gtgaaagata aatgatctgg tcatatttgc aagtatcgtt ttagagctag aaatagcaag
     4081 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tggtgctttt
     4141 tttgtttttt atgtcttcga gtcatgtaat tagttatgtc acgcttacgt tcacgccctc
     4201 caacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt
     4261 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt
     4321 ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta
     4381 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg
     4441 ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag
     4501 cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat
     4561 gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg
     4621 gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact
     4681 agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga
     4741 tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca
     4801 gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac
     4861 attttcgtat tgtttacgat tcactctatg aatagttctt actacaattt ttttgtctaa
     4921 agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag
     4981 gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag
     5041 atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt
     5101 ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg
     5161 ctctgaggtt cctatacttt ctacagaata ggaacttcgg aataggaact tcaaagcgtt
     5221 tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca
     5281 cgtcgcacct atatctgcgt gttgcctgta tatatataca tgagaagaac ggcatagtgc
     5341 gtgtttatgc ttaaatgcgt ggagggcgtg aacgtaagcg tgacataact aattacatga
     5401 ctcgaagaca taaaaaacaa aaaaagcacc accgactcgg tgccactttt tcaagttgat
     5461 aacggactag ccttatttta acttgctatt tctagctcta aaacactaat agcgaagaag
     5521 aaaagatcat ttatctttca ctgcggagaa gtttcgaacg ccgaaacatg cgcac
//
