LOCUS       sequence-462311-        9210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses estradiol-inducible CRISPR guide RNA and repair template 
            for creating a frameshift mutation in ADE2 gene of yeast..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9210)
  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 9210)
  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-ADE2
FEATURES             Location/Qualifiers
     source          1..9210
                     /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     8510..8533
                     /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 cttttgctgg ccttttgctc
     5821 gctcgcgtcg atctcccctc aagtggcaaa ctgcgactga tatgaaagta aagtatcgga
     5881 ccttctatta cggaatgtat acaaacgcta caggatacag tgatattaaa tagaaaacaa
     5941 gtctacaatt aaatggcaga aagaagaacc agcttactac taaccaccat actcatcaat
     6001 acccagagca tcggtgaaca tctgctcaaa ttcgaagtca gccatatcca gcgcaccgta
     6061 aggtgctgaa tcatgtggag tgaagccagg ccctggagag tccccgtcac ccaacatatc
     6121 taaatcaaaa tcgtctaaag cgtcagcgtg ggccatggca acgtcttcac cgtccaaatg
     6181 cagttcgcta gatgtagacc ctgctgtcgc taggtgggat tggtcggttt cttcaacgga
     6241 ggctccgcct ctagaagttg gtgcatgcaa cctgtgagcg tccagcattt ctagtaataa
     6301 atcatacaga ggaacgacgt ttttacactt catagaatac aggtgctcca tacccttgtt
     6361 agacatatgt ctaatgtgtg acaaaattag tagtaactga gctagcctct gatgctgttg
     6421 ctgcagagtc aatcctgctt tcgccataag gtgaatcaat gtatcagtga tcttgtccaa
     6481 cactctatgt atatggtctt tctcttccag gcttttcaga gttgagctta aaaatgtata
     6541 gacaccgcta tttaacaaaa taattgattt caggcaaacg aattcttctc cttgcaaatt
     6601 catcattctg aacctggagc tagtggcaag aagcatatca aaaatctcaa ccataccttc
     6661 tacgcattta ccttggttcc tgtccaataa taaattaggt gcaaataata attttacagg
     6721 gtgctccata cttctccaca ctaatccgat catcaatatt tctaaccacg cgcattcaag
     6781 aagatgtacc tggtcatgta atgtcaagtc aacaaagccg ggaactcttt tggcccaatt
     6841 aatcatatga actaattccc tgtctgctag atttgtcaaa agacccatca tggaagcctc
     6901 ggaaaaagga cgagttgggt cgtattcact gtacaatata ggtggttcag catcaagtag
     6961 tgcggaaacc atttgatcag cggtcaaaga cagtgcaaga gaattcttct tagatctttt
     7021 gatcattaat gggcttggcc acaagttggc tgccctcatg tctccagcgc ttggatcttc
     7081 tagggttgag gctgctgccg gagtgccccc acctcctgta tggatctttg tatgtctctt
     7141 tctttcatca gatctggcga actttcttcc acaaatgtca cacgcgaacg gcttctcgcc
     7201 ggtgtgggtt ctaatgtgcg tagtcaaatg gtcacttctg ctgaagtttc tcatacagat
     7261 tctacattgg aaaggctttt ggccggtgtg aatacgaata tgcctagtca gctcgtcaga
     7321 tctgctgaat ctcctatcac aactttctac agggcaagcg tagggtctcg ttcccattgt
     7381 taattcagta aattttcgat cttgggaaga aaaaagcagt aagcgtgaaa aatctaaaag
     7441 ctgatgtagt agaagatcct attctttaac aaagattgac cttttctttt tcttcttggt
     7501 ttgagtagaa aggggaagga agaatacaag agagaggaaa aaaaggaaga taaaaagaga
     7561 gcgtgatata aatgaatata tattaaacaa gagagattgg gaaggaaagg atcaaacaaa
     7621 cccaaaaata tttcaaaaag gagagagaga ggcgagtttg gtttcaaaac ggtttattta
     7681 tttatgcaag aggacgtgga agaaaaagaa gaaggaagaa aaaaatttga aagaaaaaaa
     7741 cgcgtggcgg gtaaagaaga aaatggaaaa tagaggccgg gtgacagaga aatattgagg
     7801 gttaattgga aaatatgtta gggtgaggca tatgttttta aagggactag tttatattga
     7861 attttcaaaa attcttactt tttttttgga tggacgcaaa gaagtttaat aatcatatta
     7921 catggcatta ccaccatata catatccata tacatatcca tatctaatct tacttatatg
     7981 ttgtggaaat gtaaagagcc ccattatctt agcctaaaaa aaccttctct ttggaacttt
     8041 cagtaatacg cttaactgct cattgctata ttgaagtgcg gccgcgtggg cgtgcgtggg
     8101 cgggcgtggg cgtgcgtggg cgggcgtggg cgtgcgtggg cgtctagacc gtgcgtcctc
     8161 gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa
     8221 taaagattct acaatactag cttttatggt tatgaagagg aaaaattggc agtaacctgg
     8281 ccccacaaac cttcaaatta acgaatcaaa ttaacaacca taggatgata atgcgattag
     8341 ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg atctattaac
     8401 agatatataa atggaaaagc tgcataacca ctttaactaa tactttcaac attttcagtt
     8461 tgtattactt cttattcaaa tgtcataaaa gtatcaacaa aaaattgtta atatacctct
     8521 atactttaac gtcaaggaga aaaaactata agggtgcgca tctgatgagt ccgtgaggac
     8581 gaaacgagct agctcgtcat gcgcaccctt agcgagaggt ttatcattaa ggtcaacctc
     8641 tggatgttgt ttcggcatcc tgcattgaat ctgagttact gtctgttttc ctgggtcacg
     8701 cgtaggaggg ttttccattc gtcttgaagt cgaggacttt ggcatacgat ggaagagcgt
     8761 aacttcgttg taaagaataa ggaaatgatt ccggaagctt tggaagtaag gaaacccgtt
     8821 tcttctgacg taagggtgcg caactttggc atacgatgga aggtttcaga gctatgctgg
     8881 aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     8941 gtcggtgctt tttgatggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac
     9001 accttcgggt ggcgaatggg acttaaagaa agtggaatat tcattcatat catatttttt
     9061 ctattaactg cctggtttct tttaaatttt ttattggttg tcgacttgaa cggagtgaca
     9121 atatatatat atatatttaa taatgacatc attatctgta aatctgattc ttaatgctat
     9181 tctagttatg taagagtggt cctttccata
//
