LOCUS       sequence-462042-        6667 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 NatMX 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6667)
  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 6667)
  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-Nat
FEATURES             Location/Qualifiers
     source          1..6667
                     /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     complement(2857..2876)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2858..2876)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2886..2903)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2886..2902)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2895..2917)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3025..4144)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3025..3222)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3228..3800)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(3801..4144)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      4286..4741
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4373..4392)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4583..4604
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4872..5675)
                     /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(5676..5891)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5915..5934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6034..6056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6094..6112)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6153..6656
                     /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 ttgctcaagt agagggggta atttttcccc tttattttgt tcatacattc
     1861 ttaaattgct ttgcctctcc ttttggaaag ctatacttcg gagcactgtt gagcgaaggc
     1921 tcattagata tattttctgt cattttcctt aacccaaaaa taagggaaag ggtccaaaaa
     1981 gcgctcggac aactgttgac cgtgatccga aggactggct atacagtgtt cacaaaatag
     2041 ccaagctgaa aataatgtgt agctatgttc agttagtttg gctagcaaag atataaaagc
     2101 aggtcggaaa tatttatggg cattattatg cagagcatca acatgataaa aaaaaacagt
     2161 tgaatattcc ctcaaaaggg tgcgcatctg atgagtccgt gaggacgaaa cgagctagct
     2221 cgtcatgcgc acccttagcg agaggtttat cattaaggtc aacctctgga tgttgtttcg
     2281 gcatcctgca ttgaatctga gttactgtct gttttcctgc ggccgcgttt cagagctatg
     2341 ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca
     2401 ccgagtcggt gctttttgat ggccggcatg gtcccagcct cctcgctggc gccggctggg
     2461 caacaccttc gggtggcgaa tgggacttaa agaaagtgga atattcattc atatcatatt
     2521 ttttctatta actgcctggt ttcttttaaa ttttttattg gttgtcgact tgaacggagt
     2581 gacaatatat atatatatat ttaataatga catcattatc tgtaaatctg attcttaatg
     2641 ctattctagt tatgtaagag tggtcctttc cataaaaaaa aaaaaaagaa aaaagaattt
     2701 taggaataca atgcagcttg taagtaaaat ctggaatatt catatcgcca caacttctta
     2761 tgcttataaa agcactaatg cctgaattta tgttgaaaat atgtgtcaca aataaagaaa
     2821 ctgtgacatc tgacacattt ccacgtaccc aattcgccct atagtgagtc gtattacgcg
     2881 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     2941 aatcgccttg cagcacatcc ccctttcgcc agaattcgag 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 ggggcagggc
     3241 atgctcatgt agagcgcctg ctcgccgtcc gaggcggtgc cgtcgtacag ggcggtgtcc
     3301 aggccgcaga gggtgaaccc catccgccgg tacgcgtgga tcgccggtgc gttgacgttg
     3361 gtgacctcca gccagaggtg cccggcgccc cgctcgcggg cgaactccgt cgcgagcccc
     3421 atcaacgcgc gcccgacccc gtgcccccgg tgctccgggg cgacctcgat gtcctcgacg
     3481 gtcagccggc ggttccagcc ggagtacgag acgaccacga agcccgccag gtcgccgtcg
     3541 tccccgtacg cgacgaacgt ccgggagtcc gggtcgccgt cctccccgtc gtccgattcg
     3601 tcgtccgatt cgtcgtcggg gaacaccttg gtcaggggcg ggtccaccgg cacctcccgc
     3661 agggtgaagc cgtccccggt ggcggtgacg cggaagacgg tgtcggtggt gaaggaccca
     3721 tccagtgcct cgatggcctc ggcgtccccc gggacactgg tgcggtaccg gtaagccgtg
     3781 tcgtcaagag tggtacccat ggttgtttat gttcggatgt gatgtgagaa ctgtatccta
     3841 gcaagatttt aaaaggaagt atatgaaaga agaacctcag tggcaaatcc taacctttta
     3901 tatttctcta caggggcgcg gcgtggggac aattcaacgc gtctgtgagg ggagcgtttc
     3961 cctgctcgca ggtctgcagc gaggagccgt aatttttgct tcgcgccgtg cggccatcaa
     4021 aatgtatgga tgcaaatgat tatacatggg gatgtatggg ctaaatgtac gggcgacagt
     4081 cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag gagggtattc tgggcctcca
     4141 tgtcgctggc cgggtgaccc ggcggggacg aggcaagcta aacagatctg gcgcgcctta
     4201 attaacccgg ggatcctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4261 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4321 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4381 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4441 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4501 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4561 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4621 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4681 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4741 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagggtaat
     4801 aactgatata attaaattga agctctaatt tgtgagttta gtatacatgc atttacttat
     4861 aatacagttt tttagttttg ctggccgcat cttctcaaat atgcttccca gcctgctttt
     4921 ctgtaacgtt caccctctac cttagcatcc cttccctttg caaatagtcc tcttccaaca
     4981 ataataatgt cagatcctgt agagaccaca tcatccacgg ttctatactg ttgacccaat
     5041 gcgtctccct tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc gtaaccttca
     5101 tctcttccac ccatgtctct ttgagcaata aagccgataa caaaatcttt gtcgctcttc
     5161 gcaatgtcaa cagtaccctt agtatattct ccagtagata gggagccctt gcatgacaat
     5221 tctgctaaca tcaaaaggcc tctaggttcc tttgttactt cttctgccgc ctgcttcaaa
     5281 ccgctaacaa tacctgggcc caccacaccg tgtgcattcg taatgtctgc ccattctgct
     5341 attctgtata cacccgcaga gtactgcaat ttgactgtat taccaatgtc agcaaatttt
     5401 ctgtcttcga agagtaaaaa attgtacttg gcggataatg cctttagcgg cttaactgtg
     5461 ccctccatgg aaaaatcagt caagatatcc acatgtgttt ttagtaaaca aattttggga
     5521 cctaatgctt caactaactc cagtaattcc ttggtggtac gaacatccaa tgaagcacac
     5581 aagtttgttt gcttttcgtg catgatatta aatagcttgg cagcaacagg actaggatga
     5641 gtagcagcac gttccttata tgtagctttc gacatgattt atcttcgttt cctgcaggtt
     5701 tttgttctgt gcagttgggt taagaatact gggcaatttc atgtttcttc aacactacat
     5761 atgcgtatat ataccaatct aagtctgtgc tccttccttc gttcttcctt ctgttcggag
     5821 attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat
     5881 gatgaattga aaaggtggta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5941 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6001 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6061 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6121 ttaatgtcat gataataatg gtttcttaga cggatcgctt gcctgtaact tacacgcgcc
     6181 tcgtatcttt taatgatgga ataatttggg aatttactct gtgtttattt atttttatgt
     6241 tttgtatttg gattttagaa agtaaataaa gaaggtagaa gagttacgga atgaagaaaa
     6301 aaaaataaac aaaggtttaa aaaatttcaa caaaaagcgt actttacata tatatttatt
     6361 agacaagaaa agcagattaa atagatatac attcgattaa cgataagtaa aatgtaaaat
     6421 cacaggattt tcgtgtgtgg tcttctacac agacaagatg aaacaattcg gcattaatac
     6481 ctgagagcag gaagagcaag ataaaaggta gtatttgttg gcgatccccc tagagtcttt
     6541 tacatcttcg gaaaacaaaa actatttttt ctttaatttc tttttttact ttctattttt
     6601 aatttatata tttatattaa aaaatttaaa ttataattat ttttatagca cgtgatgaaa
     6661 aggaccc
//
