LOCUS       sequence-462310-        7267 bp    DNA     circular SYN 15-APR-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 7267)
  AUTHORS   Stuecker TN, Hood SE, Pineda JM, Lenaduwe S, Winter J, Sadhu MJ, 
            Lewis JA
  TITLE     Improved vectors for retron-mediated CRISPR-Cas9 genome editing in 
            Saccharomyces cerevisiae.
  JOURNAL   bioRxiv [Preprint]. 2024 Aug 7:2024.08.06.606807. doi: 
            10.1101/2024.08.06.606807.
  PUBMED    39149293
REFERENCE   2  (bases 1 to 7267)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Aug 7:2024.08.06.606807. doi: 
            10.1101/2024.08.06.606807."
COMMENT     Sequence Label: pCRISPEY-GAL-Hyg-ADE2
FEATURES             Location/Qualifiers
     source          1..7267
                     /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(3132..3151)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3133..3151)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3161..3178)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3161..3177)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3170..3192)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            complement(3299..4874)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(3299..3496)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3502..4530)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(3502..4530)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(4531..4874)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      4987..5442
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5074..5093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5284..5305
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5573..6376)
                     /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(6377..6592)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6616..6635)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6735..6757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6795..6813)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6854..7267,1..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 ccttctgtac cgggtcacgc gtaggagggt tttccattcg
     2461 tcttgaagtc gaggactttg gcatacgatg gaagagcgta acttcgttgt aaagaataag
     2521 gaaatgattc cggaagcttt ggaagtaagg aaacccgttt cttctgacgt aagggtgcgc
     2581 aactttggca tacgatggaa ggtttcagag ctatgctgga aacagcatag caagttgaaa
     2641 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttgatggccg
     2701 gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca ccttcgggtg gcgaatggga
     2761 cttaaagaaa gtggaatatt cattcatatc atattttttc tattaactgc ctggtttctt
     2821 ttaaattttt tattggttgt cgacttgaac ggagtgacaa tatatatata tatatttaat
     2881 aatgacatca ttatctgtaa atctgattct taatgctatt ctagttatgt aagagtggtc
     2941 ctttccataa aaaaaaaaaa aagaaaaaag aattttagga atacaatgca gcttgtaagt
     3001 aaaatctgga atattcatat cgccacaact tcttatgctt ataaaagcac taatgcctga
     3061 atttatgttg aaaatatgtg tcacaaataa agaaactgtg acatctgaca catttccacg
     3121 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3181 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3241 tcgccagtcg agagctcgtt ttcgacactg gatggcggcg ttagtatcga atcgacagca
     3301 gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc gcacttaact
     3361 tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta acatttgatt
     3421 aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg aaaacaagaa
     3481 tctttttatt gtcagtactg attattcctt tgccctcgga cgagtgctgg ggcgtcggtt
     3541 tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc
     3601 gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg
     3661 cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc
     3721 aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc
     3781 gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc
     3841 gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg
     3901 gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc
     3961 ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac
     4021 ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa
     4081 atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt
     4141 ctggctaaga tcggccgcag caatggcatc cattgcctcc gcgaccggct gcagaacagc
     4201 gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca
     4261 ataggtcagg ctctcgctga attccccaat gtcaagcact tccggaatcg ggagcgcggc
     4321 cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac
     4381 ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc
     4441 cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga
     4501 cgtcgcggtg agttcaggct ttttacccat ggttgtttat gttcggatgt gatgtgagaa
     4561 ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag tggcaaatcc
     4621 taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc gtctgtgagg
     4681 ggagcgtttc cctgctcgca ggtctgcagc gaggagccgt aatttttgct tcgcgccgtg
     4741 cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg ctaaatgtac
     4801 gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag gagggtattc
     4861 tgggcctcca tgtcgctggc cgggtgaccc ggcggggacg aggcaagcta aacagactgg
     4921 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4981 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5221 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5281 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5341 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5401 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     5461 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     5521 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     5581 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     5641 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     5701 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     5761 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     5821 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     5881 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     5941 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     6001 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     6061 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     6121 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     6181 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     6241 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     6301 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     6361 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     6421 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     6481 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     6541 ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt
     6601 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     6661 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6721 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6781 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6841 ggtttcttag acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg
     6901 aataatttgg gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga
     6961 aagtaaataa agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta
     7021 aaaaatttca acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta
     7081 aatagatata cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg
     7141 gtcttctaca cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa
     7201 gataaaaggt agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa
     7261 aactatt
//
