LOCUS       sequence-208552-        6043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hph based Yeast/E.coli shuttle vector designed to clone and express 
            Cas9 programming spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6043)
  AUTHORS   Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NG, van den 
            Broek M, Daran-Lapujade P, Pronk JT, van Maris AJ, Daran JM
  TITLE     CRISPR/Cas9: a molecular Swiss army knife for simultaneous 
            introduction of multiple genetic modifications in Saccharomyces 
            cerevisiae.
  JOURNAL   FEMS Yeast Res. 2015 Mar;15(2). pii: fov004. doi: 
            10.1093/femsyr/fov004. Epub 2015 Mar 4.
  PUBMED    25743786
REFERENCE   2  (bases 1 to 6043)
  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: "2015-03"; volume: "15(2). pii: fov004. doi"; pages: " 
            10.1093/femsyr/fov004. Epub 2015 Mar 4"
COMMENT     Sequence Label: pROS12
FEATURES             Location/Qualifiers
     source          1..6043
                     /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)."
     promoter        2434..2777
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2778..3806
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2778..3806
                     /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"
     terminator      3814..4061
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    4079..4112
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4235..4503
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        4524..4598
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4603..4622
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4847..5828
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5874..5893)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5898..5972)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(5993..6043,1..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 atctgtttag
     2401 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     2461 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     2521 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     2581 aaaaattacg gctcctcgct gcggacctgc gagcagggaa acgctcccct cacagacgcg
     2641 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     2701 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     2761 tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     2821 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     2881 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     2941 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3001 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     3061 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     3121 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     3181 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     3241 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     3301 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     3361 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     3421 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     3481 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     3541 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     3601 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     3661 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     3721 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     3781 gcactcgtcc gagggcaaag gaataatctc gagtcatgta attagttatg tcacgcttac
     3841 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     3901 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     3961 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     4021 tgcttgagaa ggttttggga cgctcgaagg ctttaacgag ctctcgagaa cccttaatat
     4081 aacttcgtat aatgtatgct atacgaagtt attaggtgat atcagatcca ctagtggcct
     4141 atgccacctt tcgagaggac gatgcccgtg tctaaatgat tcgaccagcc taagaatgtt
     4201 caacccctca ctaaagggaa caaaagctgg agcttctttg aaaagataat gtatgattat
     4261 gctttcactc atatttatac agaaacttga tgttttcttt cgagtatata caaggtgatt
     4321 acatgtacgt ttgaagtaca actctagatt ttgtagtgcc ctcttgggct agcggtaaag
     4381 gtgcgcattt tttcacaccc tacaatgttc tgttcaaaag attttggtca aacgctgtag
     4441 aagtgaaagt tggtgcgcat gtttcggcgt tcgaaacttc tccgcagtga aagataaatg
     4501 atcacttgaa gattctttag tgtgttttag agctagaaat agcaagttaa aataaggcta
     4561 gtccgttatc aacttgaaaa agtggcaccg agtcggtggt gctttttttg ttttttatgt
     4621 cttcgagtca tgtaattagt tatgtcacgc ttacgttcac gccctccacg catttaagca
     4681 taaacacgca ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata
     4741 ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc
     4801 gttttcggaa acgctttgaa gttcctattc cgaagttcct attctgtaga aagtatagga
     4861 acctcagagc gcttttgaaa accaaaagcg ctctgaagac gcactttcaa aaaaccaaaa
     4921 acgcaccgga ctgtaacgag ctactaaaat attgcgaata ccgcttccac aaacattgct
     4981 caaaagtatc tctttgctat atatctctgt gctatatccc tatataacct acccatccac
     5041 ctttcgctcc ttgaacttgc atctaaactc gacctctaca ttttttatgt ttatctctag
     5101 tattactctt tagacaaaaa aattgtagta agaactattc atagagtgaa tcgaaaacaa
     5161 tacgaaaatg taaacatttc ctatacgtag tatatagaga caaaatagaa gaaaccgttc
     5221 ataattttct gaccaatgaa gaatcatcaa cgctatcact ttctgttcac aaagtatgcg
     5281 caatccacat cggtatagaa tataatcggg gatgccttta tcttgaaaaa atgcacccgc
     5341 agcttcgcta gtaatcagta aacgcgggaa gtggagtcag gcttttttta tggaagagaa
     5401 aatagacacc aaagtagcct tcttctaacc ttaacggacc tacagtgcaa aaagttatca
     5461 agagactgca ttatagagcg cacaaaggag aaaaaaagta atctaagatg ctttgttaga
     5521 aaaatagcgc tctcgggatg catttttgta gaacaaaaaa gaagtataga ttctttgttg
     5581 gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt
     5641 ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca aaaatgaagc acagattctt
     5701 cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg taaaaatgca gctcagattc
     5761 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ctacaaaatg aagcacagat
     5821 gcttcgttgg agggcgtgaa cgtaagcgtg acataactaa ttacatgact cgaagacata
     5881 aaaaacaaaa aaagcaccac cgactcggtg ccactttttc aagttgataa cggactagcc
     5941 ttattttaac ttgctatttc tagctctaaa actcctccat agagaacgta tcgatcattt
     6001 atctttcact gcggagaagt ttcgaacgcc gaaacatgcg cac
//
