LOCUS       sequence-214965-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Cas9 programming sgRNA3 and sgRNA4 targetting HXT8 and
            HXT1 respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  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 6632)
  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: pUDR211
FEATURES             Location/Qualifiers
     source          1..6632
                     /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
     protein_bind    2346..2379
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4668..4701
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4824..5092
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5113..5187
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5192..5211
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     protein_bind    5407..5454
                     /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)."
     rep_origin      5436..6417
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(6463..6482)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(6487..6561)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(6582..6632,1..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 gcaccttcgt acgctgcagg tcgacaaccc
     2341 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctagag atccgcaggc
     2401 taaccggaac ctgtattatt tagtttatgc tacgttaaat aaagaccttt cgttcacata
     2461 actgaatgtg taatggcctt gagatttcaa gcataccaag ttggtggaga cggggtcgtt
     2521 acaaaagact ctttaagcca agatttcctt gacagccttg gcgatagcat cgccaacctc
     2581 agtggtagag ttagaaccac caaggtcacc ggttctgaca cctgcatcca agacatttct
     2641 aacagcttct tcaagagccc taccttcttc aaccaaatcc aaggataact tcaacatcat
     2701 agctgcagat aagatggtag caattgggtt aaccttgttt gctggtaaat ctggggcaga
     2761 accatgacat ggttcgtaca aaccgaatgc cttgttagtg tcaggtaggg aagctagaga
     2821 tgcagaaggt aataaaccca aagaacctgg aataacagag gcttcatcgg agataatatc
     2881 accaaacatg ttgttggtaa taacaacacc gtttagctta gttggtgatt taaccaaaat
     2941 catagcagca gagtcgatca attggtgctg aacagttaat tgtgggaact cagtcttgat
     3001 ggtttcttca acagtctttc tccacaatct ggaagaggca agcacgttag ccttgtcaag
     3061 agaccagatt ggtaatggtg ggttttgttg caatgccaag aaagcagcca ttcttgtaat
     3121 tctttgaact tcaggaacac tgtatttctc agagtcccaa gcaactccgt caccttcatc
     3181 ttcttttctt tcaccaaagt agataccacc aaccaattct ctaacgacga cgaaatcggt
     3241 accctttgca tattcaggct tcaaaggaga aagatctagt aaagaatcag aagcaaagtt
     3301 acatggtctc aagttggcgt atagacccaa ttcctttctg atcttcaata gaccttgttc
     3361 tggtctaact gcgcccgtac cccattttgg accaccaaca gcacctagta agacagcatc
     3421 ggctttctta gaggcttcta gagcttcatc tggtaaagga gtgccagtgg catcgatggc
     3481 agcacccccg atcaagtgat gttggaaatt gaacttaatt tctggacgga cttcagcaat
     3541 ggcattcaag accttaatag cttcgtcagt aacttcttta ccgacgtgat cacccggtag
     3601 gacaacgata ttcttagaca tggtataatc tgtgtagtgt gggatacttt ttacttcttc
     3661 aaataggtat caacaataaa aataaatcga agcaaatgta ggaatgcgtt aaagcagatg
     3721 tacttactta gagtacatag atatatttat ataattcaat atataaaagt atatgaagca
     3781 tctgatgttg aacctgtcat gactctaaac tggaatgggt agttatgctt ctgaaggttt
     3841 tccgttagaa aatgagtgca gggatcaggc ccggaaccgg ttttagcctg aaaggaaaag
     3901 aaaaaaatgc ggtgattact agcacgtgac tgcgctgaat tggaatccac cacaagtaca
     3961 gcgaatggca agagggaagg ggaatatatt gtgcctggcg gtagtctgct atatcaaatg
     4021 ttaaacataa ctaaatgcag tagtgaatag agtaatgtat ttctacatat gtgggtaggg
     4081 ccaatgggag cccgatgtct attgtacaga tattttcctt ttatagttaa tcaattcaac
     4141 ttggaaatca ttgagtcgat atctacttgg ataactggag ccgtggcagc gtttgaaatt
     4201 tgacgagtct tcttgttttg ttcgcgcatt ttcagtttat tcaaacggga gatggcaccg
     4261 aatccactga cttctctgtt caatttgaat ggtaatagga agaaagtaca tttcgagaaa
     4321 ttttcgccca acgtcgaaat ctcatggatg atatcctcga tacaaatgac accttcatca
     4381 cctaatttct cttcaacgat attgttgtca ttcaagatta tctcttttgg ttctttatct
     4441 tctggccttt gccacattat tctagatctc ttttggatta aagaacggat agatgcaagt
     4501 gaaggttgtc cgataacaat atatggtgat acaagtttca acaatgggta cacgtccttg
     4561 gtaagtttga tgaaaacacc agtgttcaac tcaacaagtc tcaaaagaga taacactttg
     4621 taagcctttg ctgggatctt cacagcgagc tctcgagaac ccttaatata acttcgtata
     4681 atgtatgcta tacgaagtta ttaggtgata tcagatccac tagtggccta tgccaccttt
     4741 cgagaggacg atgcccgtgt ctaaatgatt cgaccagcct aagaatgttc aacccctcac
     4801 taaagggaac aaaagctgga gcttctttga aaagataatg tatgattatg ctttcactca
     4861 tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt
     4921 tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt
     4981 ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt
     5041 ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tcgaaataat
     5101 cacataacat acgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     5161 acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc ttcgagtcat
     5221 gtaattagtt atgtcacgct tacgttcacg ccctccacgc atttaagcat aaacacgcac
     5281 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg
     5341 aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa
     5401 cgctttgaag ttcctattcc gaagttccta ttctgtagaa agtataggaa cttcagagcg
     5461 cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac
     5521 tgtaacgagc tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct
     5581 ctttgctata tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct
     5641 tgaacttgca tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt
     5701 agacaaaaaa attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt
     5761 aaacatttcc tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg
     5821 accaatgaag aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc
     5881 ggtatagaat ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag
     5941 taatcagtaa acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca
     6001 aagtagcctt cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat
     6061 tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct
     6121 ctcgggatgc atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg
     6181 ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta
     6241 gcgctctcgc gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa
     6301 tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa
     6361 aattagcgct ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgttgga
     6421 gggcgtgaac gtaagcgtga cataactaat tacatgactc gaagacataa aaaacaaaaa
     6481 aagcaccacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact
     6541 tgctatttct agctctaaaa cataataaat tcaaatgtgt tgatcattta tctttcactg
     6601 cggagaagtt tcgaacgccg aaacatgcgc ac
//
