LOCUS       sequence-462302-        9035 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 9035)
  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 9035)
  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-Nat
FEATURES             Location/Qualifiers
     source          1..9035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(183..202)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(184..202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(212..229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(212..228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(221..243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(351..1470)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(351..548)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(554..1126)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1127..1470)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      1612..2067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1699..1718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1909..1930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2198..3001)
                     /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(3002..3217)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3241..3260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3360..3382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3420..3438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3479..3982
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4019..4123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4124..4192,4193..4984)
                     /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(4342..4361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5155..5743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5644..5663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8499..8522
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 aaaaaaaaaa aaagaaaaaa gaattttagg aatacaatgc agcttgtaag taaaatctgg
       61 aatattcata tcgccacaac ttcttatgct tataaaagca ctaatgcctg aatttatgtt
      121 gaaaatatgt gtcacaaata aagaaactgt gacatctgac acatttccac gtacccaatt
      181 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
      241 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagaa
      301 ttcgagctcg ttttcgacac tggatggcgg cgttagtatc gaatcgacag cagtatagcg
      361 accagcattc acatacgatt gacgcatgat attactttct gcgcacttaa cttcgcatct
      421 gggcagatga tgtcgaggcg aaaaaaaata taaatcacgc taacatttga ttaaaataga
      481 acaactacaa tataaaaaaa ctatacaaat gacaagttct tgaaaacaag aatcttttta
      541 ttgtcagtac tgattagggg cagggcatgc tcatgtagag cgcctgctcg ccgtccgagg
      601 cggtgccgtc gtacagggcg gtgtccaggc cgcagagggt gaaccccatc cgccggtacg
      661 cgtggatcgc cggtgcgttg acgttggtga cctccagcca gaggtgcccg gcgccccgct
      721 cgcgggcgaa ctccgtcgcg agccccatca acgcgcgccc gaccccgtgc ccccggtgct
      781 ccggggcgac ctcgatgtcc tcgacggtca gccggcggtt ccagccggag tacgagacga
      841 ccacgaagcc cgccaggtcg ccgtcgtccc cgtacgcgac gaacgtccgg gagtccgggt
      901 cgccgtcctc cccgtcgtcc gattcgtcgt ccgattcgtc gtcggggaac accttggtca
      961 ggggcgggtc caccggcacc tcccgcaggg tgaagccgtc cccggtggcg gtgacgcgga
     1021 agacggtgtc ggtggtgaag gacccatcca gtgcctcgat ggcctcggcg tcccccggga
     1081 cactggtgcg gtaccggtaa gccgtgtcgt caagagtggt acccatggtt gtttatgttc
     1141 ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa
     1201 cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt
     1261 caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt
     1321 tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg
     1381 tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact
     1441 gtcaaggagg gtattctggg cctccatgtc gctggccggg tgacccggcg gggacgaggc
     1501 aagctaaaca gatctggcgc gccttaatta acccggggat cctggcgtaa tagcgaagag
     1561 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct
     1621 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1681 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1741 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1801 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1861 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1921 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1981 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2041 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     2101 cggtatttca caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg
     2161 agtttagtat acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc
     2221 tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc
     2281 cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag accacatcat
     2341 ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg
     2401 tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc
     2461 cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag
     2521 tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg
     2581 ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg
     2641 cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga
     2701 ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg
     2761 ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat
     2821 gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg
     2881 tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata
     2941 gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca
     3001 tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc
     3061 aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct
     3121 tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa
     3181 atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt gcactctcag
     3241 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3301 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3361 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3421 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgga
     3481 tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa tttgggaatt
     3541 tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta aataaagaag
     3601 gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa
     3661 aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag atatacattc
     3721 gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac
     3781 aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat
     3841 ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta ttttttcttt
     3901 aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa tttaaattat
     3961 aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg gaaatgtgcg
     4021 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4081 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4141 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4201 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4261 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4321 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4381 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4441 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4501 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4561 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4621 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4681 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4741 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4801 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     4861 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4921 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4981 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5041 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5101 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5161 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5221 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5281 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5341 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5401 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5461 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5521 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5581 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5641 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5701 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaaggcca gcaacgcggc
     5761 ctttttacgg ttcctggcct tttgctggcc ttttgctggc cttttgctcg ctcgcgtcga
     5821 tctcccctca agtggcaaac tgcgactgat atgaaagtaa agtatcggac cttctattac
     5881 ggaatgtata caaacgctac aggatacagt gatattaaat agaaaacaag tctacaatta
     5941 aatggcagaa agaagaacca gcttactact aaccaccata ctcatcaata cccagagcat
     6001 cggtgaacat ctgctcaaat tcgaagtcag ccatatccag cgcaccgtaa ggtgctgaat
     6061 catgtggagt gaagccaggc cctggagagt ccccgtcacc caacatatct aaatcaaaat
     6121 cgtctaaagc gtcagcgtgg gccatggcaa cgtcttcacc gtccaaatgc agttcgctag
     6181 atgtagaccc tgctgtcgct aggtgggatt ggtcggtttc ttcaacggag gctccgcctc
     6241 tagaagttgg tgcatgcaac ctgtgagcgt ccagcatttc tagtaataaa tcatacagag
     6301 gaacgacgtt tttacacttc atagaataca ggtgctccat acccttgtta gacatatgtc
     6361 taatgtgtga caaaattagt agtaactgag ctagcctctg atgctgttgc tgcagagtca
     6421 atcctgcttt cgccataagg tgaatcaatg tatcagtgat cttgtccaac actctatgta
     6481 tatggtcttt ctcttccagg cttttcagag ttgagcttaa aaatgtatag acaccgctat
     6541 ttaacaaaat aattgatttc aggcaaacga attcttctcc ttgcaaattc atcattctga
     6601 acctggagct agtggcaaga agcatatcaa aaatctcaac cataccttct acgcatttac
     6661 cttggttcct gtccaataat aaattaggtg caaataataa ttttacaggg tgctccatac
     6721 ttctccacac taatccgatc atcaatattt ctaaccacgc gcattcaaga agatgtacct
     6781 ggtcatgtaa tgtcaagtca acaaagccgg gaactctttt ggcccaatta atcatatgaa
     6841 ctaattccct gtctgctaga tttgtcaaaa gacccatcat ggaagcctcg gaaaaaggac
     6901 gagttgggtc gtattcactg tacaatatag gtggttcagc atcaagtagt gcggaaacca
     6961 tttgatcagc ggtcaaagac agtgcaagag aattcttctt agatcttttg atcattaatg
     7021 ggcttggcca caagttggct gccctcatgt ctccagcgct tggatcttct agggttgagg
     7081 ctgctgccgg agtgccccca cctcctgtat ggatctttgt atgtctcttt ctttcatcag
     7141 atctggcgaa ctttcttcca caaatgtcac acgcgaacgg cttctcgccg gtgtgggttc
     7201 taatgtgcgt agtcaaatgg tcacttctgc tgaagtttct catacagatt ctacattgga
     7261 aaggcttttg gccggtgtga atacgaatat gcctagtcag ctcgtcagat ctgctgaatc
     7321 tcctatcaca actttctaca gggcaagcgt agggtctcgt tcccattgtt aattcagtaa
     7381 attttcgatc ttgggaagaa aaaagcagta agcgtgaaaa atctaaaagc tgatgtagta
     7441 gaagatccta ttctttaaca aagattgacc ttttcttttt cttcttggtt tgagtagaaa
     7501 ggggaaggaa gaatacaaga gagaggaaaa aaaggaagat aaaaagagag cgtgatataa
     7561 atgaatatat attaaacaag agagattggg aaggaaagga tcaaacaaac ccaaaaatat
     7621 ttcaaaaagg agagagagag gcgagtttgg tttcaaaacg gtttatttat ttatgcaaga
     7681 ggacgtggaa gaaaaagaag aaggaagaaa aaaatttgaa agaaaaaaac gcgtggcggg
     7741 taaagaagaa aatggaaaat agaggccggg tgacagagaa atattgaggg ttaattggaa
     7801 aatatgttag ggtgaggcat atgtttttaa agggactagt ttatattgaa ttttcaaaaa
     7861 ttcttacttt ttttttggat ggacgcaaag aagtttaata atcatattac atggcattac
     7921 caccatatac atatccatat acatatccat atctaatctt acttatatgt tgtggaaatg
     7981 taaagagccc cattatctta gcctaaaaaa accttctctt tggaactttc agtaatacgc
     8041 ttaactgctc attgctatat tgaagtgcgg ccgcgtgggc gtgcgtgggc gggcgtgggc
     8101 gtgcgtgggc gggcgtgggc gtgcgtgggc gtctagaccg tgcgtcctcg tcttcaccgg
     8161 tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta
     8221 caatactagc ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc
     8281 ttcaaattaa cgaatcaaat taacaaccat aggatgataa tgcgattagt tttttagcct
     8341 tatttctggg gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa
     8401 tggaaaagct gcataaccac tttaactaat actttcaaca ttttcagttt gtattacttc
     8461 ttattcaaat gtcataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg
     8521 tcaaggagaa aaaactataa gggtgcgcat ctgatgagtc cgtgaggacg aaacgagcta
     8581 gctcgtcatg cgcaccctta gcgagaggtt tatcattaag gtcaacctct ggatgttgtt
     8641 tcggcatcct gcattgaatc tgagttactg tctgttttcc tctcgaggtt tcagagctat
     8701 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     8761 accgagtcgg tgctttttga tggccggcat ggtcccagcc tcctcgctgg cgccggctgg
     8821 gcaacacctt cgggtggcga atgggactta aagaaagtgg aatattcatt catatcatat
     8881 tttttctatt aactgcctgg tttcttttaa attttttatt ggttgtcgac ttgaacggag
     8941 tgacaatata tatatatata tttaataatg acatcattat ctgtaaatct gattcttaat
     9001 gctattctag ttatgtaaga gtggtccttt ccata
//
