LOCUS       sequence-80658-e        6201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA precursor targeted against ade6L469.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6201)
  AUTHORS   Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M
  TITLE     Implementation of the CRISPR-Cas9 system in fission yeast.
  JOURNAL   Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344.
  PUBMED    25352017
REFERENCE   2  (bases 1 to 6201)
  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: "2014-10-29"; volume: "5:5344. doi"; pages: " 
            10.1038/ncomms6344"
COMMENT     Sequence Label: pMZ286
FEATURES             Location/Qualifiers
     source          1..6201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      539..1320
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     CDS             1930..2724
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     primer_bind     3350..3372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3364..3381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3365..3381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(3431..3482)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(3587..3943)
                     /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)"
     primer_bind     complement(3980..3996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3980..3996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3993..4015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4004..4020
                     /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)."
     promoter        complement(join(4028..4034,4035..4052,4053..4058))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4073..4094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4211..4228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4382..4970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4462..4481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5141..5932,5933..6001))
                     /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     5764..5783
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6002..6106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6174..6192
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataatcg atggccaacc ttccaattca ttaaatcgag tcctccatca gatgatttac
      241 aaggttttgc atagaatccc taagtaagag aagttgttag tgatttcttc aagtttacta
      301 aaatttacat acaaatggat gatctcttct ccattgtttc ctttcttctt gcaaacgcgt
      361 tttacaaaga gatgacatgc tattttctga ttattttttt ctatagtttt ctattttgta
      421 cgtccttggg agtccgaaat gtaaaatcgg ggtatggtta gtagcggcag cgttcattat
      481 ggtatataaa agataaaaag taggggcaag cgagacagaa tgggatacaa gggcatcgtc
      541 tataattata gctaaaaatt gtattttaat ttgtattttt tgtaatttta tttctcattg
      601 ttttacttaa aatgaatgcg aattagagaa aacttcaacg aaatgtcaaa ataagctcag
      661 caaaatatac aattttaggg aaagcgatca gcaacttatt ctccgttgta gattcttttt
      721 cttcataaat tccaacaatg taactgccaa ttgatttttc tctagatgct aataaattga
      781 aactaatgaa tataataaca caggttttga taaattgtaa actcttactc aaaaaaagaa
      841 tgttaaaact aatttaacct ttttataaag gctcatggcg ttaaagagga tagtaaaaat
      901 tctacaattt tttaatttaa tgtcacaatt gtcttgcgaa tataattcta ttaattttcg
      961 ctaactgctg ttttagtact attactttca atatgcctta tatgcaactt taattcatga
     1021 agaatgcaat atttttgcaa tctttcttgt actgagcgct ttttatcatt tagtactcat
     1081 tatttaattt ttcagtaaag ggcaataaat aaatttgtag aagatgcaat gtaatctcct
     1141 ctatcctttt gctcatatgt ttatgagtat acctagtcta gaaaggcttg tatttaaaat
     1201 attattcaat aaaaattcac aatttttacg acatgtgcta ccattcactt aacttcctga
     1261 ttataaaatt ggttcgttta tactaattac ttaagtacct ttaactaaac aaaatgccta
     1321 tatatatatt aatttacaat gagtgtcaga taagtcacta tgtccgagtg gttaaggagt
     1381 tagactcgcc atcgatgagc ttagctacaa atcccactgg ctatatgtat gcatttgtgt
     1441 taaaaaagtt tgtatagatt atttaatcta ctcagcattc tttctctaaa taggaatttg
     1501 ttacttaatg gagaaaaaaa tgtttcgatt tacctagtgt atttgtttgt atactcacgt
     1561 ttaatttcaa acatccattc tatcttgtgt aatttttggc atggtgaaaa agataatcag
     1621 ccttataatc tttacaaaag taagaaattc tgtaaataag ccttaatgcc cttgctttaa
     1681 attaaaatgg ttctttttca tgataatgtt tgcactttgt gaatatattt tagatagttc
     1741 tgtgaggtat aattaagatg ttttagagac ttatacaatt ttgtctttat aaattcttaa
     1801 ttgattttac catcccagtt taactatgct tcgtcggcat ctctgcacat gtcgtgtttt
     1861 cttaccgtat tgtcctacca agaacctctt ttttgcttgg atcgaaatta aaggtttaaa
     1921 agcaaagtta tggatgctag agtatttcaa agctattcag ctagagctga ggggatgaaa
     1981 aatcccattg ccaaggaatt gttggctttg atggaagaaa agcaaagcaa cttgtcagtc
     2041 gcggtcgatt tgacgaagaa atccgaaatc ttagaattgg tagataaaat tggaccctat
     2101 gtctgtgtta tcaagacaca tattgacgtt gtcgaggatt tcgaccagga tatggtagaa
     2161 aaactggtgg ccttaggtaa aaagcatcgt tttcttatct ttgaggatcg caaattcgca
     2221 gacattggaa ataccgtcaa gctacaatat gcatctggtg tgtacaaaat tgcttcttgg
     2281 gctcatatca caaattgcca tacagtgcca ggcgagggta ttatacaagg cctcaaagaa
     2341 gttggtttac ctttgggacg tggtctcttg cttttggctg aaatgtcttc caaaggctct
     2401 ttggctactg gttcctacac agagaaaacc ttagaatggt ttgagaagca taccgatttt
     2461 tgctttggct ttatagctgg tcgtcgattt cctaaccttc aaagcgacta cataactatg
     2521 tcccctggta tcggcttgga tgttaaagga gacgggctgg gacagcaata tcgtactcct
     2581 gaagaagtga ttgtaaactg cggtagcgat atcatcattg ttggtcgtgg agtctatgga
     2641 gctggtcgta atcctgttgt cgaagccaag agatatagag aagctggttg gaaggcatat
     2701 cagcaaagac tttctcagca ttaaaaaaag actaatgtaa aatttttttg gttggttatt
     2761 gaaaaagtcg atgccttgtt tgcgtttgtt ttcctaggcg ttttatgtca gaaggcattt
     2821 agaattagta tacaagtact ctttggtaaa attttatgta gcgactaaaa tattaactat
     2881 tatagataaa caccttggga ataaaaagta atttgctata gtaatttatt aaacatgctc
     2941 ctacaacatt accacaatct tttctcttgg attgacattg aataagaaaa gagtgaattt
     3001 ttttagactt gtaatgataa ctatgtacaa agccaatgaa agatgtatgt agatgaatgt
     3061 aaaataccat gtagacaaac aagataaaac ttggttataa acattggtgt tggaacagaa
     3121 taaattagat gtcaaaaagt ttcgtcaata tcacaagctc atcgcgatgg tatgcggtgt
     3181 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     3241 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     3301 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     3361 cgttgtaaaa cgacggccag tgccaagctt gcatgcctgc aggtcgactc tagaggatcc
     3421 ccacggtacc cctgtttcgt cctcacggac tcatcagacc ggaaagcaca tccggtgaca
     3481 gggcaccacc gactcggtgc cactcacttt tttcaagttg ataacggact agccttattt
     3541 taacttgcta tttctagctc taaaaccaag gcatctgaac aatagattct tcggtacagg
     3601 ttatgttttt tggcaacagc aactattcaa atcaactgaa agttttttag aattcaacaa
     3661 cttccaatac tcaagtgtcc aagctagcaa actgtttaat aatcgtgacc caacattcct
     3721 ttcatgcgca ccctcagcgt cacataaatc aacactttac aacacctatt caaagcttct
     3781 atatgccttc gtcatttaaa tgtctaacat ttcaaaaaag tgtttgctaa tggtaaattc
     3841 ttgagttttc ttggaagcac atatataacg agtgtgactg ttctacgctc cttgctgact
     3901 agtcatcagt ctacgcatgc caactaccac caacataagc aaaaggtacc cggggtaccg
     3961 agctcgaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4021 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4081 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4801 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5041 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5101 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5161 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5221 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5281 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5341 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5401 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5461 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5521 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5581 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5641 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5701 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5761 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5821 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5881 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5941 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6001 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6061 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6121 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     6181 gtatcacgag gccctttcgt c
//
