LOCUS       sequence-220879-        5464 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 5464)
  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 5464)
  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..5464
                     /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..2668
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2669..3331
                     /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        3656..3924
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3945..4019
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4024..4043
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4089..5070
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5295..5314)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5319..5393)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5414..5464,1..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 ttttccacct agcggatgac tctttttttt tcttagcgat tggcattatc
     2581 acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata tactaaaaaa
     2641 tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag taaagcgtat
     2701 tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc tagcgataga
     2761 gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca cacaatcgca
     2821 agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg ctctggccaa
     2881 gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag acgaccatca
     2941 caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc taggggccgt
     3001 gcgtggagta aaaaggtttg gatcaggatt tgcgcctttg gatgaggcac tttccagagc
     3061 ggtggtagat ctttcgaaca ggccgtacgc agttgtcgaa cttggtttgc aaagggagaa
     3121 agtaggagat ctctcttgcg agatgatccc gcattttctt gaaagctttg cagaggctag
     3181 cagaattacc ctccacgttg attgtctgcg aggcaagaat gatcatcacc gtagtgagag
     3241 tgcgttcaag gctcttgcgg ttgccataag agaagccacc tcgcccaatg gtaccaacga
     3301 tgttccctcc accaaaggtg ttcttatgta gtgacaccga ttatttaaag ctgcagcata
     3361 cgatatatat acatgtgtat atatgtatac ctatgaatgt cagtaagtat gtatacgaac
     3421 agtatgatac tgaagatgac aaggtaatgc atcattctat acgtgtcatt ctgaacgagg
     3481 cgcgctttcc ttttttcttt ttgctttttc tttttttttc tcttgaactc gacggatcta
     3541 tgcggtgtga aataccgcac agatgcacct ttcgagagga cgatgcccgt gtctaaatga
     3601 ttcgaccagc ctaagaatgt tcaacccctc actaaaggga acaaaagctg gagcttcttt
     3661 gaaaagataa tgtatgatta tgctttcact catatttata cagaaacttg atgttttctt
     3721 tcgagtatat acaaggtgat tacatgtacg tttgaagtac aactctagat tttgtagtgc
     3781 cctcttgggc tagcggtaaa ggtgcgcatt ttttcacacc ctacaatgtt ctgttcaaaa
     3841 gattttggtc aaacgctgta gaagtgaaag ttggtgcgca tgtttcggcg ttcgaaactt
     3901 ctccgcagtg aaagataaat gatcttattt atgtgaggtg aatagtttta gagctagaaa
     3961 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgg
     4021 tgcttttttt gttttttatg tcttcgagtc atgtaattag ttatgtcacg cttacgttca
     4081 cgccctccaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
     4141 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
     4201 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag
     4261 agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc
     4321 gagagcgcta ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc
     4381 ccgagagcgc tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct
     4441 ctataatgca gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc
     4501 tactttggtg tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact
     4561 gattactagc gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct
     4621 ataccgatgt ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca
     4681 ttggtcagaa aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa
     4741 tgtttacatt ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt
     4801 tgtctaaaga gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa
     4861 gttcaaggag cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag
     4921 caaagagata cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg
     4981 ttacagtccg gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc
     5041 aaaagcgctc tgaggttcct atactttcta cagaatagga acttcggaat aggaacttca
     5101 aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca
     5161 ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg
     5221 gcatagtgcg tgtttatgct taaatgcgtg gagggcgtga acgtaagcgt gacataacta
     5281 attacatgac tcgaagacat aaaaaacaaa aaaagcacca ccgactcggt gccacttttt
     5341 caagttgata acggactagc cttattttaa cttgctattt ctagctctaa aactagtaga
     5401 agaaatagtt atcgatcatt tatctttcac tgcggagaag tttcgaacgc cgaaacatgc
     5461 gcac
//
