LOCUS       sequence-305824-        6264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting Can1-3 site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6264)
  AUTHORS   Tan J, Zhang F, Karcher D, Bock R
  TITLE     Engineering of high-precision base editors for site-specific single 
            nucleotide replacement.
  JOURNAL   Nat Commun. 2019 Jan 25;10(1):439. doi: 10.1038/s41467-018-08034-8.
  PUBMED    30683865
REFERENCE   2  (bases 1 to 6264)
  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: "Nat 
            Commun."; date: "2019-01-25"; volume: "10(1):439. doi"; pages: " 
            10.1038/s41467-018-08034-8"
COMMENT     Sequence Label: pJT303_SNR52_sgRNA_Can1-3
FEATURES             Location/Qualifiers
     source          1..6264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1048..1316
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1338..1412
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1417..1436
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      1442..1689
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1707..1726)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1708..1726)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1736..1753)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1736..1752)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1745..1767)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1893..2348
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1980..1999)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2190..2211
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2479..3282)
                     /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(3283..3498)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3522..3541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3641..3663
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3701..3719)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3760..5102
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4092..4139
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        5129..5233
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5234..5302,5303..6094)
                     /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(5452..5471)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca
     1081 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta
     1141 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca
     1201 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa
     1261 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcgacg
     1321 tccaaaattg aatgactgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1381 tatcaacttg aaaaagtggc accgagtcgg tggtgctttt tttgtttttt atgtcttcga
     1441 gtcatgtaat tagttatgtc acgcttacgt tcacgccctc cccccacatc cgctctaacc
     1501 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     1561 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     1621 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
     1681 ttaatttgcg gccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     1741 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1801 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1861 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     1921 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1981 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2041 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2101 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2161 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2221 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2281 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2341 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2401 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt
     2461 tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc
     2521 tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct
     2581 tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg
     2641 acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta
     2701 accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc
     2761 gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca
     2821 tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg
     2881 cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca
     2941 ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc
     3001 aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt
     3061 aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat
     3121 tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga
     3181 agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact
     3241 aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct
     3301 gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac
     3361 actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg
     3421 ttcggagatt accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa
     3481 aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc
     3541 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3601 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3661 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3721 ttataggtta atgtcatgat aataatggtt tcttagtatg atccaatatc aaaggaaatg
     3781 atagcattga aggatgagac taatccaatt gaggagtggc agcatataga acagctaaag
     3841 ggtagtgctg aaggaagcat acgatacccc gcatggaatg ggataatatc acaggaggta
     3901 ctagactacc tttcatccta cataaataga cgcatataag tacgcattta agcataaaca
     3961 cgcactatgc cgttcttctc atgtatatat atatacaggc aacacgcaga tataggtgcg
     4021 acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat tttcggaagc gctcgttttc
     4081 ggaaacgctt tgaagttcct attccgaagt tcctattctc tagaaagtat aggaacttca
     4141 gagcgctttt gaaaaccaaa agcgctctga agacgcactt tcaaaaaacc aaaaacgcac
     4201 cggactgtaa cgagctacta aaatattgcg aataccgctt ccacaaacat tgctcaaaag
     4261 tatctctttg ctatatatct ctgtgctata tccctatata acctacccat ccacctttcg
     4321 ctccttgaac ttgcatctaa actcgacctc tacatttttt atgtttatct ctagtattac
     4381 tctttagaca aaaaaattgt agtaagaact attcatagag tgaatcgaaa acaatacgaa
     4441 aatgtaaaca tttcctatac gtagtatata gagacaaaat agaagaaacc gttcataatt
     4501 ttctgaccaa tgaagaatca tcaacgctat cactttctgt tcacaaagta tgcgcaatcc
     4561 acatcggtat agaatataat cggggatgcc tttatcttga aaaaatgcac ccgcagcttc
     4621 gctagtaatc agtaaacgcg ggaagtggag tcaggctttt tttatggaag agaaaataga
     4681 caccaaagta gccttcttct aaccttaacg gacctacagt gcaaaaagtt atcaagagac
     4741 tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa gatgctttgt tagaaaaata
     4801 gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta tagattcttt gttggtaaaa
     4861 tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa
     4921 aattagcgct ctcgcgttgc atttttgttt tacaaaaatg aagcacagat tcttcgttgg
     4981 taaaatagcg ctttcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt
     5041 tgaaaaatta gcgctctcgc gttgcatttt tgttctacaa aatgaagcac agatgcttcg
     5101 ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5161 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5221 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5281 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5341 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5401 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5461 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5521 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5581 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5641 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5701 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5761 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5821 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5881 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5941 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6001 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6061 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6121 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6181 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6241 cgtagaaaag atcaaaggat cttc
//
