LOCUS       sequence-230325-        8996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA with BbsI placeholder for target cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8996)
  AUTHORS   Hayashi A, Tanaka K
  TITLE     Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing 
            in Fission Yeast.
  JOURNAL   G3 (Bethesda). 2019 Feb 12. pii: g3.118.200976. doi: 
            10.1534/g3.118.200976.
  PUBMED    30755408
REFERENCE   2  (bases 1 to 8996)
  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). 2019 Feb 12. pii: g3.118.200976. doi: 
            10.1534/g3.118.200976."
COMMENT     Sequence Label: pAH233
FEATURES             Location/Qualifiers
     source          1..8996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(722..1816)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1817..2224)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        3437..3793
                     /label=rrk1 promoter/leader
                     /note="promoter and leader RNA for the Schizosaccharomyces 
                     pombe rrk1 gene encoding the K-RNA component of RNase P 
                     (Jacobs et al., 2014)"
     misc_RNA        3900..3951
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     rep_origin      4992..5775
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(6125..6142)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6125..6141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6134..6156)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6354..6809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6441..6460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6651..6672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6826..6845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6945..6967
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7005..7023)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7091..7195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7196..7264,7265..8056)
                     /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(7414..7433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8227..8815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8716..8735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8969..8986
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 tcgatcgact acgtcgttaa ggccgtttct gacagagtaa aattcttgag ggaactttca
      301 ccattatggg aaatggttca agaaggtatt gacttaaact ccatcaaatg gtcaggtcat
      361 tgagtgtttt ttatttgttg tatttttttt tttttagaga aaatcctcca atatataaat
      421 taggaatcat agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      481 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      541 aatttgctta cctgtattcc tttacatcct cctttttctc cttcttgata aatgtatgta
      601 gattgcgtat atagtttcgt ctaccctatg aacatattcc attttgtaat ttcgtgtcgt
      661 ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt
      721 tttaagcaag gattttctta acttcttcgg cgacagcatc accgacttcc gtggtactgt
      781 tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt
      841 caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca
      901 agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg
      961 gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca
     1021 acaaacccaa ggaacctggg ataacggagg cttcatcgga gatgatatca ccaaacatgt
     1081 tgctggtgat tataatacca tttaggtggg ttgggttctt aactaggatc atggcggcag
     1141 aatcaatcaa ttgatgttga accttcaatg tagggaattc gttcttgatg gtttcctcca
     1201 cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag
     1261 gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt
     1321 ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcatct tcctttctct
     1381 taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa
     1441 attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta
     1501 agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac
     1561 taccggtacc ccatttagga ccacccacag cacctaacaa aacggcatca accttcttgg
     1621 aggcttccag cgcctcatct ggaagtggga cacctgtagc atcgatagca gcaccaccaa
     1681 ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa
     1741 ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct
     1801 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat attgctgaaa
     1861 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
     1921 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
     1981 cgcttctcta ttctatatga aaagccggtt ccgcggctct cacctttcct ttttctccca
     2041 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc
     2101 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa
     2161 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac
     2221 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac
     2281 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac
     2341 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt
     2401 tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta gaagttctcc
     2461 tcgacaagct tgcatgcctg cagggaaatc ataacatagt ataataggaa tcatatgaca
     2521 tttactcttg catagcaata tcctcttctt cgccatcagc aatccgacga gcatatgctt
     2581 cagcctcttg caaaagctca tcaggaacgg gtgtatgaat accgtttgtt tcaacaccaa
     2641 cccaagtcat ctcaatttca tgctctttgt tgttttcttc gtcatgagtt gcgtataatc
     2701 taaaaaagag tgaattatta gcaaatagaa tgaaaaaaaa ttgtactcaa caaagcaaaa
     2761 cgtacattct agcagcttct ttaaccgcat ctttcatttt taatgaggaa aaattcagtt
     2821 tttctaattc gtttcgagct acttgccgac ctttaccagc tgcagcaccg ttgtaacccc
     2881 aataaacacc attaggttca accatataca aatgtggacc ttctgagtca taagtagcaa
     2941 caaaagacat aacaccgaat ggacggacgc tagaatagca agtgaaaagt tgaacgtagt
     3001 tccccaagcg atcagcaata actgttccag ggatgggaga tccgtagtta tctctccatg
     3061 aggtggcttc gtcccttgca cgttttacga tgtgttggcc atcaggaata aagccagtag
     3121 tagcaatacc aatgtgtcga tcgaccgacc caattctgtt atttactcgc ggcttaagta
     3181 gtttgctagt aactaccttt tctagagcta gaataactcc atcttcacat tttattccaa
     3241 tgcaagtact aaatacaatg ttaaaatcaa tcgataagga aattcaatcc accttacctt
     3301 gcattttcga cggctttata cgcgtactcc gcttggaata aacgaccatc cggggagaag
     3361 aataatccta aatcgtatcc agtaccaatt gaactctgtt tgttaatgtg atgtgctact
     3421 ccttgctaac ttaccatttg cttatgttgg tggtagttgg catgcgtaga ctgatgacta
     3481 gtcagcaagg agcgtagaac agtcacactc gttatatatg tgcttccaag aaaactcaag
     3541 aatttaccat tagcaaacac ttttttgaaa tgttagacat ttaaatgacg aaggcatata
     3601 gaagctttga ataggtgttg taaagtgttg atttatgtga cgctgagggt gcgcatgaaa
     3661 ggaatgttgg gtcacgatta ttaaacagtt tgctagcttg gacacttgag tattggaagt
     3721 tgttgaattc taaaaaactt tcagttgatt tgaatagttg ctgttgccaa aaaacataac
     3781 ctgtaccaaa gaacaccggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt
     3841 aaaataaggc tagtccgtta tcaacttgaa aaaagtgagt ggcaccgagt cggtggtgcc
     3901 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag gtacggttat
     3961 ttgttagcaa tggtaggaga aaattaggga ttgtttacca aaacaacgtg aacaaatagt
     4021 tatagtttta atttaactca tgaacatatt atcggaaaaa atctactcat ttatgtctgg
     4081 gtttagctcc ctttatggtt tcgttttgca gtagaataca ctatggtatt tattttgtgc
     4141 actacaaacc gtgcttttta tgtgaacgtt ttgatgaaaa cgttctagag attgatttat
     4201 tattcaatgt taaagtaact cattttgcta tatgtatctt tttttcagcg aactttcgtt
     4261 actgagcagt tcaagcagtg atttggcatt atggtatcaa taaagacata tcattaaggc
     4321 aaggctacgg aaagaaaaaa gaaggttttt aaattaatat acaacaaatt aaccgtgatc
     4381 ggacagtata tttgatgcaa agatataatg aaagttggga aaataagaat atagcactca
     4441 aatgattgcc ttacaaaagc attgctttct ctacaggtaa ggttgaagtg cctttacggt
     4501 atcttcaaaa accgcatcgg gatcttgctc ggcatcgatg gttatcagac gattttgtga
     4561 tttcagatat tcgacgacag gcatgctggc tttagtatat gtaacaaagc gtttcttgat
     4621 cgactcaatg ttgtcatcag aacggccact agtcttacca cgatgaataa gacgcttcaa
     4681 catggtttcc tgaccacaac ggaaataaag agcaaactta gcggggcaga ccgacttttc
     4741 aaaaccttcg cattgatcca tctcacgggg gaaaccgtca atcaagaact tgtcaattcc
     4801 tttatcatgg cattccttca ttttagtctc taaaagggat atagttattt ccatgggcac
     4861 aattttacca tctttgatat attctttaat aaggtttccg tatttgctgc caggacgcga
     4921 attcgagtct aactccttaa ccactcggac atagtgactt atctgacact cattgtaaat
     4981 taatatatat ataggcattt tgtttagtta aaggtactta agtaattagt ataaacgaac
     5041 caattttata atcaggaagt taagtgaatg gtagcacatg tcgtaaaaat tgtgaatttt
     5101 tattgaataa tattttaaat acaagccttt ctagactagg tatactcata aacatatatg
     5161 agcaaaagga tagaggagat tacattgcat cttctacaaa tttatttatt gccctttact
     5221 gaaaaattaa ataatgagta ctaaatgata aaaagcgctc agtacaagaa agattgcaaa
     5281 aatattgcat tcttcatgaa ttaaagttgc atataaggca tattgaaagt aatagtacta
     5341 aaacagcagt tagcgaaaat taatagaatt atattcgcaa gacaattgtg acattaaatt
     5401 aaaaaattgt agaattttta ctatcctctt taacgccatg agcctttata aaaaggttaa
     5461 attagtttta acattctttt tttgagtaag agtttacaat ttatcaaaac ctgtgttatt
     5521 atattcatta gtttcaattt attagcatct agagaaaaat caattggcag ttacattgtt
     5581 ggaatttatg aagaaaaaga atctacaacg gagaataagt tgctgatcgc tttccctaaa
     5641 attgtatatt ttgctgagct tattttgaca tttcgttgaa gttttctcta attcgcattc
     5701 attttaagta aaacaatgag aaataaaatt acaaaaaata caaattaaaa tacaattttt
     5761 agctataatt atagacgatg cccttgtatc ccattctgtc tcgcttgccc ctacttttta
     5821 tcttttatat accataatga acgctgccgc tactaaccat accccgattt tacatttcgg
     5881 actcccaagg acgtacaaaa tagaaaacta tagaaaaaaa taatcagaaa atagcatgtc
     5941 atctctttgt aaaacgcgtt tgcaagaaga aaggaaacaa tggagaagag atcatccatt
     6001 tgtatgtaaa ttttagtaaa cttgaagaaa tcactaacaa cttctcttac ttagggattc
     6061 tatgcaaaac cttgtaaatc atctgatgga ggactcgatt taatgaattg gaaggttgga
     6121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     6181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     6241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     6301 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     6361 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     6421 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6481 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6541 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     6601 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6661 gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat
     6721 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6781 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     6841 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     6901 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6961 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     7021 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     7081 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     7141 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     7201 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     7261 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     7321 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     7381 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     7441 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     7501 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     7561 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     7621 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7681 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     7741 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7801 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7861 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7921 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7981 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     8041 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     8101 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     8161 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     8221 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     8281 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     8341 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     8401 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     8461 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     8521 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     8581 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     8641 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     8701 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     8761 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     8821 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     8881 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     8941 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
