LOCUS       sequence-462307-        6865 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 KanMX 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6865)
  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 6865)
  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
FEATURES             Location/Qualifiers
     source          1..6865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(119..187,188..979)
                     /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(337..356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1150..1738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1639..1658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2845..2864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2846..2864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2874..2891)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2874..2890)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2883..2905)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            3003..4359
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        3003..3346
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3347..4156
                     /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(3414..3433)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4162..4359
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      4472..4927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4559..4578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4769..4790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5058..5861)
                     /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(5862..6077)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6101..6120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6220..6242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6280..6298)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6339..6842
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
       61 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      661 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1021 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1081 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1141 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1201 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1261 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1321 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1381 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1441 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1501 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1561 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1621 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1681 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1741 ggccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctggcctttt
     1801 gctcaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt
     1861 gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata
     1921 ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa
     1981 ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa
     2041 taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata
     2101 tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct
     2161 caaaagggtg cgcatctgat gagtccgtga ggacgaaacg agctagctcg tcatgcgcac
     2221 ccttagcgag aggtttatca ttaaggtcaa cctctggatg ttgtttcggc atcctgcatt
     2281 gaatctgagt tactgtctgt tttcctgcgg ccgcgtttca gagctatgct ggaaacagca
     2341 tagcaagttg aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2401 tttttgatgg ccggcatggt cccagcctcc tcgctggcgc cggctgggca acaccttcgg
     2461 gtggcgaatg ggacttaaag aaagtggaat attcattcat atcatatttt ttctattaac
     2521 tgcctggttt cttttaaatt ttttattggt tgtcgacttg aacggagtga caatatatat
     2581 atatatattt aataatgaca tcattatctg taaatctgat tcttaatgct attctagtta
     2641 tgtaagagtg gtcctttcca taaaaaaaaa aaaaagaaaa aagaatttta ggaatacaat
     2701 gcagcttgta agtaaaatct ggaatattca tatcgccaca acttcttatg cttataaaag
     2761 cactaatgcc tgaatttatg ttgaaaatat gtgtcacaaa taaagaaact gtgacatctg
     2821 acacatttcc acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     2881 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2941 gcacatcccc ctttcgccag tctgtttagc ttgcctcgtc cccgccgggt cacccggcca
     3001 gcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat
     3061 gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca
     3121 tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg
     3181 agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga
     3241 gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa
     3301 tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg gtaaggaaaa
     3361 gactcacgtt tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     3421 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     3481 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     3541 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     3601 tatccgtact cctgatgatg catggttact caccactgcg atccccggca aaacagcatt
     3661 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     3721 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     3781 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     3841 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc
     3901 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     3961 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     4021 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     4081 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     4141 cgatgagttt ttctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca
     4201 tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat
     4261 attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata
     4321 tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc
     4381 cgccatccag tgtcgaaaac gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4441 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
     4501 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4561 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4621 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4681 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4741 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4801 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4861 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     4921 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag
     4981 ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt
     5041 acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct
     5101 gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt
     5161 ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga
     5221 cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa
     5281 ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg
     5341 ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat
     5401 gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc
     5461 ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat
     5521 tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca
     5581 aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta
     5641 actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt
     5701 ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa
     5761 gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta
     5821 ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg
     5881 caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca
     5941 ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt
     6001 tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa
     6061 aaaaatgatg aattgaaaag gtggtatggt gcactctcag tacaatctgc tctgatgccg
     6121 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6181 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6241 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6301 tataggttaa tgtcatgata ataatggttt cttagacgga tcgcttgcct gtaacttaca
     6361 cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt
     6421 ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga
     6481 agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata
     6541 tttattagac aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg
     6601 taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat
     6661 taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga
     6721 gtcttttaca tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct
     6781 atttttaatt tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg
     6841 atgaaaagga cccaggtggc acttt
//
