LOCUS       sequence-373961-        7816 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of a single-spacer CRISPR array with spacer #1 targeting 
            the mRNA of deGFP and expression of PbuCas13b in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7816)
  AUTHORS   Wandera KG, Alkhnbashi OS, Bassett HVI, Mitrofanov A, Hauns S, Migur
            A, Backofen R, Beisel CL
  TITLE     Anti-CRISPR prediction using deep learning reveals an inhibitor of 
            Cas13b nucleases.
  JOURNAL   Mol Cell. 2022 May 24. pii: S1097-2765(22)00437-3. doi: 
            10.1016/j.molcel.2022.05.003.
  PUBMED    35649413
REFERENCE   2  (bases 1 to 7816)
  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: "Mol Cell. 
            2022 May 24. pii: S1097-2765(22)00437-3. doi: 
            10.1016/j.molcel.2022.05.003."
COMMENT     Sequence Label: pPlacPbuCas13b-gRNA-T-deGFP
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(236..338)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(730..747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(864..1409)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(944..963)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(1521..1526,1527..1543,1544..1549)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        join(1699..1704,1705..1722,1723..1728)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        5128..5162
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     5204..5225
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5520..5539)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(join(7392..7816,1..235))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
       61 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
      121 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
      181 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
      241 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
      301 cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt
      361 tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat
      421 cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga
      481 tttagtgtat gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctgtccc
      541 tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc
      601 tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc
      661 atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata
      721 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat
      781 ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt
      841 gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa
      901 atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      961 ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
     1021 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
     1081 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
     1141 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
     1201 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
     1261 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
     1321 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
     1381 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
     1441 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     1501 ataagttgta attctcatgt ttgacagctt atcatcgata agctttaatg cggtagttta
     1561 tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc
     1621 gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg
     1681 ccgggcctct tgcgggattt tacactttat gcttccggct cgtatgttag gaggtcttta
     1741 tcatgcaaaa acaagataaa ttatttgtag atagaaagaa aaatgctata tttgcctttc
     1801 ctaaatatat aacaataatg gaaaacaaag agaaacctga accaatttat tatgaattga
     1861 cagataagca tttctgggct gcctttctta acttggcgcg ccacaatgtc tataccacca
     1921 ttaatcacat caacagacgt ttggaaatag cagagctaaa agacgatgga tatatgatgg
     1981 gcattaaagg cagctggaac gagcaagcga aaaaactcga caaaaaagtt cggctgagag
     2041 acttgatcat gaaacacttt ccttttctgg aagctgcagc ttatgaaatg acaaactcta
     2101 aaagtcccaa caataaggag caacgggaaa aggaacaatc cgaagcccta tcacttaaca
     2161 acttgaagaa tgttctattc attttcttgg aaaaacttca ggttctcagg aactactact
     2221 cccattacaa gtattctgaa gaatccccaa aaccgatatt tgaaacaagc cttttgaaaa
     2281 acatgtataa ggttttcgat gccaatgtgc ggttagtaaa gagagactat atgcatcatg
     2341 agaatataga catgcaaaga gattttacac atctcaacag gaaaaaacaa gtgggacgaa
     2401 caaaaaacat catagacagt cctaactttc attaccattt tgcagataaa gaaggcaata
     2461 tgaccatagc aggcttactg ttcttcgtct ctttatttct tgataaaaaa gacgctattt
     2521 ggatgcagaa aaagctaaag ggatttaaag atggcagaaa cttgcgtgag caaatgacaa
     2581 acgaagtatt ttgcagaagc cgtatatctt tgcctaagct aaaattagag aatgtacaga
     2641 caaaagactg gatgcaactt gatatgctta acgaattggt tcgctgtccc aaatcgctgt
     2701 atgaacggtt acgagaaaaa gatagggaaa gttttaaagt acctttcgat attttctctg
     2761 acgactataa tgctgaagaa gaaccattta agaacacgct tgtaaggcat caagaccgct
     2821 ttccatattt tgtcttgaga tatttcgatt taaatgagat attcgaacaa ctccgcttcc
     2881 aaatagattt aggtacctat catttctcta tctataacaa acgtatcggt gacgaagatg
     2941 aagttcgcca tctcacacat catctatatg gctttgcgcg cattcaggac tttgcaccac
     3001 aaaaccaacc agaagaatgg agaaaattag tgaaagactt agatcatttt gagacatcgc
     3061 aagagcctta catatctaag actgctccac attaccattt agagaacgaa aagataggca
     3121 ttaagttttg cagtgcccac aacaacctct ttccatctct tcagaccgac aaaacatgca
     3181 atggaagaag taaatttaat ctcggaacac aatttacagc agaagcattt ttaagtgtcc
     3241 acgaattgtt gccgatgatg ttttattatc tgttattgac caaggattac tccagaaaag
     3301 aaagcgcaga caaagtagag ggaattataa gaaaagaaat aagcaacata tacgctatct
     3361 acgatgcttt tgccaataat gagataaata gcattgcaga cttaacccgt cggcttcaaa
     3421 atacaaatat actacaaggc catttgccaa aacaaatgat ttccatattg aaaggcagac
     3481 aaaaagatat gggaaaagag gctgagcgca aaattgggga aatgatagac gacacccaaa
     3541 gaagacttga cttgctctgc aagcagacaa atcagaagat aaggattggc aaaaggaatg
     3601 ccggtctgct aaaatcggga aaaattgcag attggttggt aaatgacatg atgcgcttcc
     3661 aacctgttca aaaagaccaa aataacattc caataaacaa tagcaaggct aacagcaccg
     3721 aatatcgaat gcttcagcgt gcgcttgccc ttttcggcag tgagaacttc cgccttaagg
     3781 catattttaa tcaaatgaat cttgtgggga atgacaatcc gcatcctttc cttgcagaaa
     3841 cacaatggga acatcaaact aacattctgt ctttctaccg aaactatttg gaagcgagga
     3901 aaaagtatct taagggctta aaaccacaaa actggaaaca atatcagcat tttctcatac
     3961 ttaaagtaca gaagaccaac cgcaacacat tagttacagg atggaaaaac agtttcaatc
     4021 tcccccgtgg aatatttacc caaccaataa gggaatggtt tgagaaacat aataattcaa
     4081 aaagaatata cgaccaaata ctatcttttg accgagtagg gtttgttgca aaagctattc
     4141 ccttatactt tgcagaagaa tataaagaca atgtacagcc tttctacgat tatccgttca
     4201 atatcggcaa cagacttaaa ccaaaaaaaa ggcaattctt agataaaaaa gagcgtgtcg
     4261 aattgtggca aaagaataaa gagctattca aaaattatcc gtctgaaaag aaaaagacag
     4321 acttagctta tcttgatttt ttatcatgga aaaagttcga acgcgaactg cgacttatca
     4381 agaatcagga tatcgtcaca tggttgatgt tcaaagaact attcaatatg gctactgtgg
     4441 aaggattgaa gattggagaa atacatctga gggacatcga tacaaacacg gcaaatgagg
     4501 aaagcaacaa tattctaaat agaatcatgc ccatgaagtt gcctgttaaa acatacgaaa
     4561 cagacaataa aggaaatata ttaaaggaaa gacctttagc aaccttttat atcgaagaaa
     4621 cggaaaccaa agtcctgaaa cagggcaatt tcaaagcatt ggtaaaagac cgaagactca
     4681 atgggctatt ctcatttgca gaaactacag acttgaactt agaggaacat cctatcagca
     4741 aattgagtgt agaccttgag cttataaaat atcagactac aaggattagt atctttgaga
     4801 tgacattagg actggaaaag aaactcatag acaaatattc cacactacct acagatagct
     4861 tcagaaatat gcttgaaagg tggttgcagt gcaaagctaa tagaccggaa ttaaagaatt
     4921 acgttaatag tttgattgca gtgagaaatg ctttttccca caaccagtat cccatgtatg
     4981 atgccacact ctttgctgaa gttaaaaaat ttactctttt cccttctgtc gatacaaaaa
     5041 agattgagct aaatatagcg ccacagttac tggaaatagt aggaaaagcc ataaaagaaa
     5101 ttgaaaaatc agaaaataaa aactaaattg acagctagct cagtcctagg tataatgcta
     5161 gcgctgagtt gcatctgcct tctttttgaa aggtaaaaac aaccgtcgcc gtccagctcg
     5221 accaggatgg gaagttgcat ctgccttctt tttgaaaggt aaaaacaaca tcgtccattc
     5281 cgacagcatc gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct
     5341 atgcgcaccc gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc
     5401 ttcgctactt ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtggat
     5461 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     5521 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     5581 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     5641 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     5701 cttcctaatg caggagtcgc ataagggaga gcgtcgaccg atgcccttga gagccttcaa
     5761 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     5821 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     5881 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     5941 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     6001 ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg cgttcgcgac
     6061 gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca tcgggatgcc
     6121 cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac agcttcaagg
     6181 atcgctcgcg gctcttacca gcctaacttc gatcattgga ccgctgatcg tcacggcgat
     6241 ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg ccgccctata
     6301 ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat
     6361 ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc caatcaattc
     6421 ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat cgcgtccgcc
     6481 atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc
     6541 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     6601 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     6661 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     6721 aagtccccta cgtgctgctg aagttgcccg caacagagag tggaaccaac cggtgatacc
     6781 acgatactat gactgagagt caacgccatg agcggcctca tttcttattc tgagttacaa
     6841 cagtccgcac cgctgccggt agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     6901 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgcccaaca
     6961 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgccc tgcaccatta
     7021 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     7081 taacgaagcg ctaaccgttt ttatcaggct ctgggaggca gaataaatga tcatatcgtc
     7141 aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac
     7201 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     7261 ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc
     7321 atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca ccaataactg
     7381 ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     7441 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     7501 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     7561 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     7621 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     7681 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     7741 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     7801 atcaccagct caccgt
//
