LOCUS       sequence-461835-        6842 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 6842)
  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 6842)
  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-ADE2
FEATURES             Location/Qualifiers
     source          1..6842
                     /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(3133..3152)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3134..3152)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3162..3179)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3162..3178)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3171..3193)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3301..4420)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(3301..3498)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3504..4076)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(4077..4420)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      4562..5017
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4649..4668)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4859..4880
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5148..5951)
                     /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(5952..6167)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6191..6210)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6310..6332
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6370..6388)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6429..6842,1..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 tgttttcctt ttggttcgtt ccgggtcacg cgtaggaggg ttttccattc
     2461 gtcttgaagt cgaggacttt ggcatacgat ggaagagcgt aacttcgttg taaagaataa
     2521 ggaaatgatt ccggaagctt tggaagtaag gaaacccgtt tcttctgacg taagggtgcg
     2581 caactttggc atacgatgga aggtttcaga gctatgctgg aaacagcata gcaagttgaa
     2641 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttgatggcc
     2701 ggcatggtcc cagcctcctc gctggcgccg gctgggcaac accttcgggt ggcgaatggg
     2761 acttaaagaa agtggaatat tcattcatat catatttttt ctattaactg cctggtttct
     2821 tttaaatttt ttattggttg tcgacttgaa cggagtgaca atatatatat atatatttaa
     2881 taatgacatc attatctgta aatctgattc ttaatgctat tctagttatg taagagtggt
     2941 cctttccata aaaaaaaaaa aaagaaaaaa gaattttagg aatacaatgc agcttgtaag
     3001 taaaatctgg aatattcata tcgccacaac ttcttatgct tataaaagca ctaatgcctg
     3061 aatttatgtt gaaaatatgt gtcacaaata aagaaactgt gacatctgac acatttccac
     3121 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     3181 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3241 ttcgccagaa ttcgagctcg ttttcgacac tggatggcgg cgttagtatc gaatcgacag
     3301 cagtatagcg accagcattc acatacgatt gacgcatgat attactttct gcgcacttaa
     3361 cttcgcatct gggcagatga tgtcgaggcg aaaaaaaata taaatcacgc taacatttga
     3421 ttaaaataga acaactacaa tataaaaaaa ctatacaaat gacaagttct tgaaaacaag
     3481 aatcttttta ttgtcagtac tgattagggg cagggcatgc tcatgtagag cgcctgctcg
     3541 ccgtccgagg cggtgccgtc gtacagggcg gtgtccaggc cgcagagggt gaaccccatc
     3601 cgccggtacg cgtggatcgc cggtgcgttg acgttggtga cctccagcca gaggtgcccg
     3661 gcgccccgct cgcgggcgaa ctccgtcgcg agccccatca acgcgcgccc gaccccgtgc
     3721 ccccggtgct ccggggcgac ctcgatgtcc tcgacggtca gccggcggtt ccagccggag
     3781 tacgagacga ccacgaagcc cgccaggtcg ccgtcgtccc cgtacgcgac gaacgtccgg
     3841 gagtccgggt cgccgtcctc cccgtcgtcc gattcgtcgt ccgattcgtc gtcggggaac
     3901 accttggtca ggggcgggtc caccggcacc tcccgcaggg tgaagccgtc cccggtggcg
     3961 gtgacgcgga agacggtgtc ggtggtgaag gacccatcca gtgcctcgat ggcctcggcg
     4021 tcccccggga cactggtgcg gtaccggtaa gccgtgtcgt caagagtggt acccatggtt
     4081 gtttatgttc ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat
     4141 gaaagaagaa cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt
     4201 ggggacaatt caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg
     4261 agccgtaatt tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata
     4321 catggggatg tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca
     4381 cgtcaagact gtcaaggagg gtattctggg cctccatgtc gctggccggg tgacccggcg
     4441 gggacgaggc aagctaaaca gatctggcgc gccttaatta acccggggat cctggcgtaa
     4501 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4561 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4621 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4681 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4741 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4801 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4861 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4921 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4981 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     5041 cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta aattgaagct
     5101 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     5161 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     5221 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     5281 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     5341 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     5401 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     5461 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     5521 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     5581 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     5641 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     5701 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     5761 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     5821 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     5881 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     5941 gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag
     6001 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     6061 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     6121 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt
     6181 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     6241 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6301 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6361 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6421 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
     6481 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
     6541 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
     6601 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
     6661 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
     6721 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
     6781 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
     6841 tt
//
