LOCUS       sequence-462314-        9165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast CEN plasmid for estradiol-inducible expression of CRISPR guide
            RNA and repair template for homologous recombination, with Z3 
            promoter and Z3EV transcription factor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9165)
  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 9165)
  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-Z3-Kan
FEATURES             Location/Qualifiers
     source          1..9165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(131..199,200..991)
                     /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(349..368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4506..4529
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     complement(5225..5244)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5226..5244)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5254..5271)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5254..5270)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5263..5285)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(5352..6708)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(5352..5549)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(5555..6364)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6278..6297
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6365..6708)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      6784..7239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6871..6890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7081..7102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7370..8173)
                     /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(8174..8389)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(8413..8432)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8532..8554
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8592..8610)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    8651..9154
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
       61 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      361 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
      661 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
      721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
      781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
      901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
      961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aaggccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1801 tgctggcctt ttgctcgctc gcgtcgatct cccctcaagt ggcaaactgc gactgatatg
     1861 aaagtaaagt atcggacctt ctattacgga atgtatacaa acgctacagg atacagtgat
     1921 attaaataga aaacaagtct acaattaaat ggcagaaaga agaaccagct tactactaac
     1981 caccatactc atcaataccc agagcatcgg tgaacatctg ctcaaattcg aagtcagcca
     2041 tatccagcgc accgtaaggt gctgaatcat gtggagtgaa gccaggccct ggagagtccc
     2101 cgtcacccaa catatctaaa tcaaaatcgt ctaaagcgtc agcgtgggcc atggcaacgt
     2161 cttcaccgtc caaatgcagt tcgctagatg tagaccctgc tgtcgctagg tgggattggt
     2221 cggtttcttc aacggaggct ccgcctctag aagttggtgc atgcaacctg tgagcgtcca
     2281 gcatttctag taataaatca tacagaggaa cgacgttttt acacttcata gaatacaggt
     2341 gctccatacc cttgttagac atatgtctaa tgtgtgacaa aattagtagt aactgagcta
     2401 gcctctgatg ctgttgctgc agagtcaatc ctgctttcgc cataaggtga atcaatgtat
     2461 cagtgatctt gtccaacact ctatgtatat ggtctttctc ttccaggctt ttcagagttg
     2521 agcttaaaaa tgtatagaca ccgctattta acaaaataat tgatttcagg caaacgaatt
     2581 cttctccttg caaattcatc attctgaacc tggagctagt ggcaagaagc atatcaaaaa
     2641 tctcaaccat accttctacg catttacctt ggttcctgtc caataataaa ttaggtgcaa
     2701 ataataattt tacagggtgc tccatacttc tccacactaa tccgatcatc aatatttcta
     2761 accacgcgca ttcaagaaga tgtacctggt catgtaatgt caagtcaaca aagccgggaa
     2821 ctcttttggc ccaattaatc atatgaacta attccctgtc tgctagattt gtcaaaagac
     2881 ccatcatgga agcctcggaa aaaggacgag ttgggtcgta ttcactgtac aatataggtg
     2941 gttcagcatc aagtagtgcg gaaaccattt gatcagcggt caaagacagt gcaagagaat
     3001 tcttcttaga tcttttgatc attaatgggc ttggccacaa gttggctgcc ctcatgtctc
     3061 cagcgcttgg atcttctagg gttgaggctg ctgccggagt gcccccacct cctgtatgga
     3121 tctttgtatg tctctttctt tcatcagatc tggcgaactt tcttccacaa atgtcacacg
     3181 cgaacggctt ctcgccggtg tgggttctaa tgtgcgtagt caaatggtca cttctgctga
     3241 agtttctcat acagattcta cattggaaag gcttttggcc ggtgtgaata cgaatatgcc
     3301 tagtcagctc gtcagatctg ctgaatctcc tatcacaact ttctacaggg caagcgtagg
     3361 gtctcgttcc cattgttaat tcagtaaatt ttcgatcttg ggaagaaaaa agcagtaagc
     3421 gtgaaaaatc taaaagctga tgtagtagaa gatcctattc tttaacaaag attgaccttt
     3481 tctttttctt cttggtttga gtagaaaggg gaaggaagaa tacaagagag aggaaaaaaa
     3541 ggaagataaa aagagagcgt gatataaatg aatatatatt aaacaagaga gattgggaag
     3601 gaaaggatca aacaaaccca aaaatatttc aaaaaggaga gagagaggcg agtttggttt
     3661 caaaacggtt tatttattta tgcaagagga cgtggaagaa aaagaagaag gaagaaaaaa
     3721 atttgaaaga aaaaaacgcg tggcgggtaa agaagaaaat ggaaaataga ggccgggtga
     3781 cagagaaata ttgagggtta attggaaaat atgttagggt gaggcatatg tttttaaagg
     3841 gactagttta tattgaattt tcaaaaattc ttactttttt tttggatgga cgcaaagaag
     3901 tttaataatc atattacatg gcattaccac catatacata tccatataca tatccatatc
     3961 taatcttact tatatgttgt ggaaatgtaa agagccccat tatcttagcc taaaaaaacc
     4021 ttctctttgg aactttcagt aatacgctta actgctcatt gctatattga agtgcggccg
     4081 cgtgggcgtg cgtgggcggg cgtgggcgtg cgtgggcggg cgtgggcgtg cgtgggcgtc
     4141 tagaccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt gcctcgcgcc
     4201 gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg aagaggaaaa
     4261 attggcagta acctggcccc acaaaccttc aaattaacga atcaaattaa caaccatagg
     4321 atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc gaagcgatga
     4381 tttttgatct attaacagat atataaatgg aaaagctgca taaccacttt aactaatact
     4441 ttcaacattt tcagtttgta ttacttctta ttcaaatgtc ataaaagtat caacaaaaaa
     4501 ttgttaatat acctctatac tttaacgtca aggagaaaaa actataaggg tgcgcatctg
     4561 atgagtccgt gaggacgaaa cgagctagct cgtcatgcgc acccttagcg agaggtttat
     4621 cattaaggtc aacctctgga tgttgtttcg gcatcctgca ttgaatctga gttactgtct
     4681 gttttcctct cgaggtttca gagctatgct ggaaacagca tagcaagttg aaataaggct
     4741 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttgatgg ccggcatggt
     4801 cccagcctcc tcgctggcgc cggctgggca acaccttcgg gtggcgaatg ggacttaaag
     4861 aaagtggaat attcattcat atcatatttt ttctattaac tgcctggttt cttttaaatt
     4921 ttttattggt tgtcgacttg aacggagtga caatatatat atatatattt aataatgaca
     4981 tcattatctg taaatctgat tcttaatgct attctagtta tgtaagagtg gtcctttcca
     5041 taaaaaaaaa aaaaagaaaa aagaatttta ggaatacaat gcagcttgta agtaaaatct
     5101 ggaatattca tatcgccaca acttcttatg cttataaaag cactaatgcc tgaatttatg
     5161 ttgaaaatat gtgtcacaaa taaagaaact gtgacatctg acacatttcc acgtacccaa
     5221 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     5281 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5341 aattcgagct ccagtatagc gaccagcatt cacatacgat tgacgcatga tattactttc
     5401 tgcgcactta acttcgcatc tgggcagatg atgtcgaggc gaaaaaaaat ataaatcacg
     5461 ctaacatttg attaaaatag aacaactaca atataaaaaa actatacaaa tgacaagttc
     5521 ttgaaaacaa gaatcttttt attgtcagta ctgattagaa aaactcatcg agcatcaaat
     5581 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     5641 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     5701 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     5761 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     5821 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     5881 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     5941 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     6001 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     6061 tgccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     6121 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     6181 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     6241 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     6301 ataaatcagc atccatgttg gaatttaatc gcggcctcga aacgtgagtc ttttccttac
     6361 ccatggttgt ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg
     6421 aagtatatga aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg
     6481 cgcggcgtgg ggacaattca acgcgtctgt gaggggagcg tttccctgct cgcaggtctg
     6541 cagcgaggag ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa
     6601 tgattataca tggggatgta tgggctaaat gtacgggcga cagtcacatc atgcccctga
     6661 gctgcgcacg tcaagactgt caaggagggt attctgggcc tccatgtcgg atcctggcgt
     6721 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     6781 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     6841 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     6901 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     6961 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     7021 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7081 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7141 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     7201 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     7261 tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag
     7321 ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct
     7381 ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct
     7441 tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag
     7501 agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac
     7561 ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt
     7621 gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag
     7681 tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc
     7741 taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca
     7801 ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt
     7861 actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat
     7921 tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca
     7981 agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca
     8041 gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca
     8101 tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg
     8161 tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta
     8221 agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa
     8281 gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc
     8341 aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg
     8401 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     8461 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     8521 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     8581 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     8641 ttcttagacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
     8701 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
     8761 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
     8821 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
     8881 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
     8941 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
     9001 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
     9061 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
     9121 aatttaaatt ataattattt ttatagcacg tgatgaaaag gaccc
//
