LOCUS       sequence-462309-        7027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses galactose-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 7027)
  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 7027)
  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-Kan-ADE2
FEATURES             Location/Qualifiers
     source          1..7027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        127..231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(232..300,301..1092)
                     /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(450..469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1263..1851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1752..1771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3120..3139)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3121..3139)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3149..3166)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3149..3165)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3158..3180)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            3278..4634
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        3278..3621
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3622..4431
                     /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     complement(3689..3708)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4437..4634
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      4747..5202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4834..4853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5044..5065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5333..6136)
                     /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(6137..6352)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6376..6395)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6495..6517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6555..6573)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6614..7027,1..90)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
       61 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
      121 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      181 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      241 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      301 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      361 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      421 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      481 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      541 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      601 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      661 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      721 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      781 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      841 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      901 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
      961 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1021 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1081 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1141 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1201 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1261 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1321 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1381 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1441 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1501 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1561 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1621 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1681 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1741 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1801 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaaggccagc
     1861 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctggcct tttgctcaag
     1921 tagagggggt aatttttccc ctttattttg ttcatacatt cttaaattgc tttgcctctc
     1981 cttttggaaa gctatacttc ggagcactgt tgagcgaagg ctcattagat atattttctg
     2041 tcattttcct taacccaaaa ataagggaaa gggtccaaaa agcgctcgga caactgttga
     2101 ccgtgatccg aaggactggc tatacagtgt tcacaaaata gccaagctga aaataatgtg
     2161 tagctatgtt cagttagttt ggctagcaaa gatataaaag caggtcggaa atatttatgg
     2221 gcattattat gcagagcatc aacatgataa aaaaaaacag ttgaatattc cctcaaaagg
     2281 gtgcgcatct gatgagtccg tgaggacgaa acgagctagc tcgtcatgcg cacccttagc
     2341 gagaggttta tcattaaggt caacctctgg atgttgtttc ggcatcctgc attgaatctg
     2401 agttactgtc tgttttcctg ggtcacgcgt aggagggttt tccattcgtc ttgaagtcga
     2461 ggactttggc atacgatgga agagcgtaac ttcgttgtaa agaataagga aatgattccg
     2521 gaagctttgg aagtaaggaa acccgtttct tctgacgtaa gggtgcgcaa ctttggcata
     2581 cgatggaagg tttcagagct atgctggaaa cagcatagca agttgaaata aggctagtcc
     2641 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt gatggccggc atggtcccag
     2701 cctcctcgct ggcgccggct gggcaacacc ttcgggtggc gaatgggact taaagaaagt
     2761 ggaatattca ttcatatcat attttttcta ttaactgcct ggtttctttt aaatttttta
     2821 ttggttgtcg acttgaacgg agtgacaata tatatatata tatttaataa tgacatcatt
     2881 atctgtaaat ctgattctta atgctattct agttatgtaa gagtggtcct ttccataaaa
     2941 aaaaaaaaaa gaaaaaagaa ttttaggaat acaatgcagc ttgtaagtaa aatctggaat
     3001 attcatatcg ccacaacttc ttatgcttat aaaagcacta atgcctgaat ttatgttgaa
     3061 aatatgtgtc acaaataaag aaactgtgac atctgacaca tttccacgta cccaattcgc
     3121 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3181 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagtctgt
     3241 ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac atggaggccc agaataccct
     3301 ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc cgtacattta
     3361 gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc gcacggcgcg
     3421 aagcaaaaat tacggctcct cgctgcagac ctgcgagcag ggaaacgctc ccctcacaga
     3481 cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc
     3541 actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca gttctcacat
     3601 cacatccgaa cataaacaac catgggtaag gaaaagactc acgtttcgag gccgcgatta
     3661 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     3721 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     3781 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     3841 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     3901 ttactcacca ctgcgatccc cggcaaaaca gcattccagg tattagaaga atatcctgat
     3961 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     4021 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     4081 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     4141 gaacaagtct ggaaagaaat gcataagctt ttgccattct caccggattc agtcgtcact
     4201 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     4261 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     4321 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     4381 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagtactg
     4441 acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag
     4501 ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct
     4561 gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc
     4621 tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgtcg aaaacgctgg
     4681 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4741 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4801 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4861 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4921 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4981 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5041 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5101 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5161 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     5221 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     5281 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     5341 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     5401 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     5461 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     5521 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     5581 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     5641 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     5701 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     5761 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     5821 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     5881 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     5941 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     6001 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     6061 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     6121 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     6181 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     6241 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     6301 ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt
     6361 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     6421 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6481 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6541 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6601 ggtttcttag acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg
     6661 aataatttgg gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga
     6721 aagtaaataa agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta
     6781 aaaaatttca acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta
     6841 aatagatata cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg
     6901 gtcttctaca cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa
     6961 gataaaaggt agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa
     7021 aactatt
//
