LOCUS       sequence-461834-        7093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast CEN plasmid for galactose-inducible expression of CRISPR guide
            RNA and repair template for homologous recombination, with HygMX 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7093)
  AUTHORS   Stuecker TN, Hood SE, Molina Pineda J, Lenaduwe S, Winter J, Sadhu 
            MJ, Lewis JA
  TITLE     Improved vectors for retron-mediated CRISPR-Cas9 genome editing in 
            Saccharomyces cerevisiae.
  JOURNAL   G3 (Bethesda). 2025 Aug 4:jkaf175. doi: 10.1093/g3journal/jkaf175.
  PUBMED    40758833
REFERENCE   2  (bases 1 to 7093)
  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: "G3 
            (Bethesda). 2025 Aug 4:jkaf175. doi: 10.1093/g3journal/jkaf175."
COMMENT     Sequence Label: pCRISPEY-GAL-Hyg
FEATURES             Location/Qualifiers
     source          1..7093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(132..200,201..992)
                     /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     complement(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2858..2877)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2859..2877)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2887..2904)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2887..2903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2896..2918)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3025..4600)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(3025..3222)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3228..4256)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(3228..4256)
                     /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"
     promoter        complement(4257..4600)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      4713..5168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4800..4819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5010..5031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5299..6102)
                     /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(6103..6318)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6342..6361)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6461..6483
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6521..6539)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6580..7083
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
       61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
      541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
      601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
      661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
      721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
      781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
      841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
      901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
      961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1741 gcctatggaa aaaggccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     1801 ttgctggcct tttgctcaag tagagggggt aatttttccc ctttattttg ttcatacatt
     1861 cttaaattgc tttgcctctc cttttggaaa gctatacttc ggagcactgt tgagcgaagg
     1921 ctcattagat atattttctg tcattttcct taacccaaaa ataagggaaa gggtccaaaa
     1981 agcgctcgga caactgttga ccgtgatccg aaggactggc tatacagtgt tcacaaaata
     2041 gccaagctga aaataatgtg tagctatgtt cagttagttt ggctagcaaa gatataaaag
     2101 caggtcggaa atatttatgg gcattattat gcagagcatc aacatgataa aaaaaaacag
     2161 ttgaatattc cctcaaaagg gtgcgcatct gatgagtccg tgaggacgaa acgagctagc
     2221 tcgtcatgcg cacccttagc gagaggttta tcattaaggt caacctctgg atgttgtttc
     2281 ggcatcctgc attgaatctg agttactgtc tgttttcctg cggccgcgtt tcagagctat
     2341 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     2401 accgagtcgg tgctttttga tggccggcat ggtcccagcc tcctcgctgg cgccggctgg
     2461 gcaacacctt cgggtggcga atgggactta aagaaagtgg aatattcatt catatcatat
     2521 tttttctatt aactgcctgg tttcttttaa attttttatt ggttgtcgac ttgaacggag
     2581 tgacaatata tatatatata tttaataatg acatcattat ctgtaaatct gattcttaat
     2641 gctattctag ttatgtaaga gtggtccttt ccataaaaaa aaaaaaaaga aaaaagaatt
     2701 ttaggaatac aatgcagctt gtaagtaaaa tctggaatat tcatatcgcc acaacttctt
     2761 atgcttataa aagcactaat gcctgaattt atgttgaaaa tatgtgtcac aaataaagaa
     2821 actgtgacat ctgacacatt tccacgtacc caattcgccc tatagtgagt cgtattacgc
     2881 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2941 taatcgcctt gcagcacatc cccctttcgc cagtcgagag ctcgttttcg acactggatg
     3001 gcggcgttag tatcgaatcg acagcagtat agcgaccagc attcacatac gattgacgca
     3061 tgatattact ttctgcgcac ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa
     3121 aatataaatc acgctaacat ttgattaaaa tagaacaact acaatataaa aaaactatac
     3181 aaatgacaag ttcttgaaaa caagaatctt tttattgtca gtactgatta ttcctttgcc
     3241 ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg
     3301 gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat
     3361 cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc
     3421 aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat
     3481 cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac
     3541 cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg
     3601 ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa
     3661 tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg
     3721 agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca
     3781 tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc
     3841 ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcaat ggcatccatt
     3901 gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg
     3961 acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca
     4021 agcacttccg gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg
     4081 tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag
     4141 ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac
     4201 ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt acccatggtt
     4261 gtttatgttc ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat
     4321 gaaagaagaa cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt
     4381 ggggacaatt caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg
     4441 agccgtaatt tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata
     4501 catggggatg tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca
     4561 cgtcaagact gtcaaggagg gtattctggg cctccatgtc gctggccggg tgacccggcg
     4621 gggacgaggc aagctaaaca gactggcgta atagcgaaga ggcccgcacc gatcgccctt
     4681 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     4741 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4801 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4861 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4921 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4981 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5041 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5101 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5161 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5221 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt
     5281 tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc
     5341 tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct
     5401 tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg
     5461 acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta
     5521 accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc
     5581 gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca
     5641 tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg
     5701 cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca
     5761 ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc
     5821 aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt
     5881 aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat
     5941 tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga
     6001 agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact
     6061 aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct
     6121 gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac
     6181 actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg
     6241 ttcggagatt accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa
     6301 aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc
     6361 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6421 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6481 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     6541 ttataggtta atgtcatgat aataatggtt tcttagacgg atcgcttgcc tgtaacttac
     6601 acgcgcctcg tatcttttaa tgatggaata atttgggaat ttactctgtg tttatttatt
     6661 tttatgtttt gtatttggat tttagaaagt aaataaagaa ggtagaagag ttacggaatg
     6721 aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact ttacatatat
     6781 atttattaga caagaaaagc agattaaata gatatacatt cgattaacga taagtaaaat
     6841 gtaaaatcac aggattttcg tgtgtggtct tctacacaga caagatgaaa caattcggca
     6901 ttaatacctg agagcaggaa gagcaagata aaaggtagta tttgttggcg atccccctag
     6961 agtcttttac atcttcggaa aacaaaaact attttttctt taatttcttt ttttactttc
     7021 tatttttaat ttatatattt atattaaaaa atttaaatta taattatttt tatagcacgt
     7081 gatgaaaagg acc
//
