LOCUS       sequence-208539-        6007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli-S. cerevisiae shuttle plasmid harbors gRNA expression 
            cassettes targeting S. cerevisiae chromosomal X-3 site (HXK1 gene). 
            Can function as a gRNA expression empty backbone plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6007)
  AUTHORS   Wang X, Yang J, Yang S, Jiang Y
  TITLE     Unraveling the genetic basis of fast l-arabinose consumption on top 
            of recombinant xylose-fermenting Saccharomyces cerevisiae.
  JOURNAL   Biotechnol Bioeng. 2018 Sep 10. doi: 10.1002/bit.26827.
  PUBMED    30199094
REFERENCE   2  (bases 1 to 6007)
  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: "Biotechnol 
            Bioeng. 2018 Sep 10. doi: 10.1002/bit.26827."
COMMENT     Sequence Label: pYES2-gRNA-hyg-MCS
FEATURES             Location/Qualifiers
     source          1..6007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(682..1025)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        1032..1300
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1321..1395
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1400..1419
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1586..1603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1757..2345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1837..1856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2516..3307,3308..3376))
                     /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     3139..3158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4077..4954
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     complement(5160..5181)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     gene            complement(join(5457..6007,1..1025))
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(5457..5654)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(join(5660..6007,1..681))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(join(5660..6007,1..681))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
ORIGIN
        1 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
       61 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
      121 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
      181 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
      241 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
      301 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc
      361 ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag
      421 gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa
      481 gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac
      541 atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg
      601 catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt
      661 gagttcaggc tttttaccca tggttgttta tgttcggatg tgatgtgaga actgtatcct
      721 agcaagattt taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt
      781 atatttctct acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt
      841 ccctgctcgc aggtccgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca
      901 aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag
      961 tcacatcatg cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc
     1021 atgtcactag ttctttgaaa agataatgta tgattatgct ttcactcata tttatacaga
     1081 aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg aagtacaact
     1141 ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt cacaccctac
     1201 aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt
     1261 tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc aatttcatcc atcaattcct
     1321 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     1381 ggcaccgagt cggtggtgct ttttttgttt tttatgtcta gatcctagga tccaagcttg
     1441 gtaccgagct cgtcgacgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg
     1501 catctagagg gccgcaggcc ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3361 aatgttgaat actcatactc ttcctttttc aatgggtaat aactgatata attaaattga
     3421 agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt tttgaaaagc
     3481 tagcttatcg atgataagct gtcaaacatg agaattaatt ccacggacta tagactatac
     3541 ctagtatact ccgtctactg tacgatacac ttccgctcag gtccttgtcc tttaacgagg
     3601 ccttaccact cttttgttac tctattgatc cagctcagca aaggcagtgt gatctaagat
     3661 tctatcttcg cgatgtagta aaactagcta gaccgagaaa gagactagaa atgcaaaagg
     3721 cacttctaca atggctgcca tcattattat ccgatgtgac gctgcagctt ctcaatgata
     3781 ttcgaatacg ctttgaggag atacagccta atatccgaca aactgtttta cagatttacg
     3841 atcgtacttg ttacccatca ttgaattttg aacatccgaa cctgggagtt ttccctgaaa
     3901 cagatagtat atttgaacct gtataataat atatagtcta gcgctttacg gaagacaatg
     3961 tatgtatttc ggttcctgga gaaactattg catctattgc ataggtaatc ttgcacgtcg
     4021 catccccggt tcattttctg cgtttccatc ttgcacttca atagcatatc tttgttaacg
     4081 aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa
     4141 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc
     4201 aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt
     4261 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt
     4321 ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta
     4381 tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt
     4441 ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc
     4501 tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga
     4561 agctgcgggt gcattttttc aagataaagg catccccgat tatattctat accgatgtgg
     4621 attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa
     4681 ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt
     4741 cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt
     4801 aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg
     4861 aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca aagagatact
     4921 tttgagcaat gtttgtggaa gcggtattcg caatgggaag ctccaccccg gttgataatc
     4981 agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta agcgttaata
     5041 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5101 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5161 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5221 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5281 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5341 ggggaaagcc ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg
     5401 tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag agatcccagt
     5461 atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc acttaacttc
     5521 gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac atttgattaa
     5581 aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa aacaagaatc
     5641 tttttattgt cagtactgat tattcctttg ccctcggacg agtgctgggg cgtcggtttc
     5701 cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga
     5761 tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg
     5821 cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa
     5881 tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc ctccgctcga
     5941 agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga
     6001 cctcgta
//
