LOCUS       sequence-234835-        8994 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 8994)
  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 8994)
  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..8994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        107..158
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     rep_origin      1199..1982
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(2332..2349)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2332..2348)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2341..2363)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2561..3016
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2648..2667)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2858..2879
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3033..3052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3152..3174
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3212..3230)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3298..3402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3403..3471,3472..4263)
                     /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(3621..3640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4434..5022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4923..4942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5176..5193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5310..5331
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5346..5351,5352..5369,5370..5376)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5384..5400
                     /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     5389..5411
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5408..5424
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5408..5424
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(5925..7019)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(7020..7425)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa ataaggctag
       61 tccgttatca acttgaaaaa agtgagtggc accgagtcgg tggtgccctg tcaccggatg
      121 tgctttccgg tctgatgagt ccgtgaggac gaaacaggta cggttatttg ttagcaatgg
      181 taggagaaaa ttagggattg tttaccaaaa caacgtgaac aaatagttat agttttaatt
      241 taactcatga acatattatc ggaaaaaatc tactcattta tgtctgggtt tagctccctt
      301 tatggtttcg ttttgcagta gaatacacta tggtatttat tttgtgcact acaaaccgtg
      361 ctttttatgt gaacgttttg atgaaaacgt tctagagatt gatttattat tcaatgttaa
      421 agtaactcat tttgctatat gtatcttttt ttcagcgaac tttcgttact gagcagttca
      481 agcagtgatt tggcattatg gtatcaataa agacatatca ttaaggcaag gctacggaaa
      541 gaaaaaagaa ggtttttaaa ttaatataca acaaattaac cgtgatcgga cagtatattt
      601 gatgcaaaga tataatgaaa gttgggaaaa taagaatata gcactcaaat gattgcctta
      661 caaaagcatt gctttctcta caggtaaggt tgaagtgcct ttacggtatc ttcaaaaacc
      721 gcatcgggat cttgctcggc atcgatggtt atcagacgat tttgtgattt cagatattcg
      781 acgacaggca tgctggcttt agtatatgta acaaagcgtt tcttgatcga ctcaatgttg
      841 tcatcagaac ggccactagt cttaccacga tgaataagac gcttcaacat ggtttcctga
      901 ccacaacgga aataaagagc aaacttagcg gggcagaccg acttttcaaa accttcgcat
      961 tgatccatct cacgggggaa accgtcaatc aagaacttgt caattccttt atcatggcat
     1021 tccttcattt tagtctctaa aagggatata gttatttcca tgggcacaat tttaccatct
     1081 ttgatatatt ctttaataag gtttccgtat ttgctgccag gacgcgaatt cgagtctaac
     1141 tccttaacca ctcggacata gtgacttatc tgacactcat tgtaaattaa tatatatata
     1201 ggcattttgt ttagttaaag gtacttaagt aattagtata aacgaaccaa ttttataatc
     1261 aggaagttaa gtgaatggta gcacatgtcg taaaaattgt gaatttttat tgaataatat
     1321 tttaaataca agcctttcta gactaggtat actcataaac atatatgagc aaaaggatag
     1381 aggagattac attgcatctt ctacaaattt atttattgcc ctttactgaa aaattaaata
     1441 atgagtacta aatgataaaa agcgctcagt acaagaaaga ttgcaaaaat attgcattct
     1501 tcatgaatta aagttgcata taaggcatat tgaaagtaat agtactaaaa cagcagttag
     1561 cgaaaattaa tagaattata ttcgcaagac aattgtgaca ttaaattaaa aaattgtaga
     1621 atttttacta tcctctttaa cgccatgagc ctttataaaa aggttaaatt agttttaaca
     1681 ttcttttttt gagtaagagt ttacaattta tcaaaacctg tgttattata ttcattagtt
     1741 tcaatttatt agcatctaga gaaaaatcaa ttggcagtta cattgttgga atttatgaag
     1801 aaaaagaatc tacaacggag aataagttgc tgatcgcttt ccctaaaatt gtatattttg
     1861 ctgagcttat tttgacattt cgttgaagtt ttctctaatt cgcattcatt ttaagtaaaa
     1921 caatgagaaa taaaattaca aaaaatacaa attaaaatac aatttttagc tataattata
     1981 gacgatgccc ttgtatccca ttctgtctcg cttgccccta ctttttatct tttatatacc
     2041 ataatgaacg ctgccgctac taaccatacc ccgattttac atttcggact cccaaggacg
     2101 tacaaaatag aaaactatag aaaaaaataa tcagaaaata gcatgtcatc tctttgtaaa
     2161 acgcgtttgc aagaagaaag gaaacaatgg agaagagatc atccatttgt atgtaaattt
     2221 tagtaaactt gaagaaatca ctaacaactt ctcttactta gggattctat gcaaaacctt
     2281 gtaaatcatc tgatggagga ctcgatttaa tgaattggaa ggttggaatt cactggccgt
     2341 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2401 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2461 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     2521 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     2581 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     2641 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2701 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2761 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2821 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2881 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     2941 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3001 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3061 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3121 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3181 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3241 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3301 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3361 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3421 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3481 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3541 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3601 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3661 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3721 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3781 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3841 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3901 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3961 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4021 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4081 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4141 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4201 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4261 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4321 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4381 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4441 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4501 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4561 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4621 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4681 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4741 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4801 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4861 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4921 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4981 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5041 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5101 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5161 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     5221 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5281 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     5341 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     5401 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgtcgatcg actacgtcgt
     5461 taaggccgtt tctgacagag taaaattctt gagggaactt tcaccattat gggaaatggt
     5521 tcaagaaggt attgacttaa actccatcaa atggtcaggt cattgagtgt tttttatttg
     5581 ttgtattttt ttttttttag agaaaatcct ccaatatata aattaggaat catagtttca
     5641 tgattttctg ttacacctaa ctttttgtgt ggtgccctcc tccttgtcaa tattaatgtt
     5701 aaagtgcaat tctttttcct tatcacgttg agccattagt atcaatttgc ttacctgtat
     5761 tcctttacat cctccttttt ctccttcttg ataaatgtat gtagattgcg tatatagttt
     5821 cgtctaccct atgaacatat tccattttgt aatttcgtgt cgtttctatt atgaatttca
     5881 tttataaagt ttatgtacaa atatcataaa aaaagagaat ctttttaagc aaggattttc
     5941 ttaacttctt cggcgacagc atcaccgact tcggtggtac tgttggaacc acctaaatca
     6001 ccagttctga tacctgcatc caaaaccttt ttaactgcat cttcaatggc cttaccttct
     6061 tcaggcaagt tcaatgacaa tttcaacatc attgcagcag acaagatagt ggcgataggg
     6121 ttgaccttat tctttggcaa atctggagca gaaccgtggc atggttcgta caaaccaaat
     6181 gcggtgttct tgtctggcaa agaggccaag gacgcagatg gcaacaaacc caaggaacct
     6241 gggataacgg aggcttcatc ggagatgata tcaccaaaca tgttgctggt gattataata
     6301 ccatttaggt gggttgggtt cttaactagg atcatggcgg cagaatcaat caattgatgt
     6361 tgaaccttca atgtagggaa ttcgttcttg atggtttcct ccacagtttt tctccataat
     6421 cttgaagagg ccaaaacatt agctttatcc aaggaccaaa taggcaatgg tggctcatgt
     6481 tgtagggcca tgaaagcggc cattcttgtg attctttgca cttctggaac ggtgtattgt
     6541 tcactatccc aagcgacacc atcaccatca tcttcctttc tcttaccaaa gtaaatacct
     6601 cccactaatt ctctgacaac aacgaagtca gtacctttag caaattgtgg cttgattgga
     6661 gataagtcta aaagagagtc ggatgcaaag ttacatggtc ttaagttggc gtacaattga
     6721 agttctttac ggatttttag taaaccttgt tcaggtctaa cactaccggt accccattta
     6781 ggaccaccca cagcacctaa caaaacggca tcagccttct tggaggcttc cagcgcctca
     6841 tctggaagtg gaacacctgt agcatcgata gcagcaccac caattaaatg attttcgaaa
     6901 tcgaacttga cattggaacg aacatcagaa atagctttaa gaaccttaat ggcttcggct
     6961 gtgatttctt gaccaacgtg gtcacctggc aaaacgacga tcttcttagg ggcagacatt
     7021 agaatggtat atccttgaaa tatatatata tattgctgaa atgtaaaagg taagaaaagt
     7081 tagaaagtaa gacgattgct aaccacctat tggaaaaaac aataggtcct taaataatat
     7141 tgtcaacttc aagtattgtg atgcaagcat ttagtcatga acgcttctct attctatatg
     7201 aaaagccggt tccggcgctc tcacctttcc tttttctccc aatttttcag ttgaaaaagg
     7261 tatatgcgtc aggcgacctc tgaaattaac aaaaaatttc cagtcatcga atttgattct
     7321 gtgcgatagc gcccctgtgt gttctcgtta tgttgaggaa aaaaataatg gttgctaaga
     7381 gattcgaact cttgcatctt acgatacctg agtattccca cagttaactg cggtcaagat
     7441 atttcttgaa tcaggcgcct tagaccgctc ggccaaacaa ccaattactt gttgagaaat
     7501 agagtataat tatcctataa atataacgtt tttgaacaca catgaacaag gaagtacagg
     7561 acaattgatt ttgaagagaa tgtggatttt gatgtaattg ttgggattcc atttttaata
     7621 aggcaataat attaggtatg tagatatact agaagttctc ctcgacaagc ttgcatgcct
     7681 gcagggaaat cataacatag tataatagga atcatatgac atttactctt gcatagcaat
     7741 atcctcttct tcgccatcag caatccgacg agcatatgct tcagcctctt gcaaaagctc
     7801 atcaggaacg ggtgtatgaa taccgtttgt ttcaacacca acccaagtca tctcaatttc
     7861 atgctctttg ttgttttctt cgtcatgagt tgcgtataat ctaaaaaaga gtgaattatt
     7921 agcaaataga atgaaaaaaa attgtactca acaaagcaaa acgtacattc tagcagcttc
     7981 tttaaccgca tctttcattt ttaatgagga aaaattcagt ttttctaatt cgtttcgagc
     8041 tacttgccga cctttaccag ctgcagcacc gttgtaaccc caataaacac cattaggttc
     8101 aaccatatac aaatgtggac cttctgagtc ataagtagca acaaaagaca taacaccgaa
     8161 tggacggacg ctagaatagc aagtgaaaag ttgaacgtag ttccccaagc gatcagcaat
     8221 aactgttcca gggatgggag atccgtagtt atctctccat gaggtggctt cgtcccttgc
     8281 acgttttacg atgtgttggc catcaggaat aaagccagta gtagcaatac caatgtgtcg
     8341 atcgaccgac ccaattctgt tatttactcg cggcttaagt agtttgctag taactacctt
     8401 ttctagagct agaataactc catcttcaca ttttattcca atgcaagtac taaatacaat
     8461 gttaaaatca atcgataagg aaattcaatc caccttacct tgcattttcg acggctttat
     8521 acgcgtactc cgcttggaat aaacgaccat ccggggagaa gaataatcct aaatcgtatc
     8581 cagtaccaat tgaactctgt ttgttaatgt gatgtgctac tccttgctaa cttaccattt
     8641 gcttatgttg gtggtagttg gcatgcgtag actgatgact agtcagcaag gagcgtagaa
     8701 cagtcacact cgttatatat gtgcttccaa gaaaactcaa gaatttacca ttagcaaaca
     8761 cttttttgaa atgttagaca tttaaatgac gaaggcatat agaagctttg aataggtgtt
     8821 gtaaagtgtt gatttatgtg acgctgaggg tgcgcatgaa aggaatgttg ggtcacgatt
     8881 attaaacagt ttgctagctt ggacacttga gtattggaag ttgttgaatt ctaaaaaact
     8941 ttcagttgat ttgaatagtt gctgttgcca aaaaacataa cctgtaccaa agaa
//
