LOCUS       sequence-325301-        3448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of single-spacer CRISPR array with spacer #2
            targeting deGFP mRNA for LsCas13a in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3448)
  AUTHORS   Wandera KG, Beisel CL
  TITLE     Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free 
            Transcription-Translation Systems.
  JOURNAL   Methods Mol Biol. 2022;2404:135-153. doi: 
            10.1007/978-1-0716-1851-6_7.
  PUBMED    34694607
REFERENCE   2  (bases 1 to 3448)
  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: "Methods Mol
            Biol."; date: "2022"; volume: "2404:135-153. doi"; pages: " 
            10.1007/978-1-0716-1851-6_7"
COMMENT     Sequence Label: pLsCas13a-gRNA-2
FEATURES             Location/Qualifiers
     source          1..3448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..45
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(231..248)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      401..487
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      579..606
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        625..716
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(717..785,786..1577)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(935..954)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1619..2074
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1706..1725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1916..1937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      2185..2773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2674..2693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2927..2944
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2959..3099
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3025..3044)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3185..3207
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3345..3364
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat ttgacagcta gctcagtcct aggtataatg ctagcgctag cgaattcgag
       61 ctcggtacca agacgtcaac tcgaccctga tatagaccac cccaatatcg aaggggacta
      121 aaacggtcgg ggtagcggct gaagcactgc acgccggata tagaccaccc caatatcgaa
      181 ggggactaaa acaagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag
      241 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg
      301 gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt
      361 gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca
      421 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
      481 gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
      541 aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg
      601 cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc
      661 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
      721 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
      781 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
      841 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
      901 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
      961 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1021 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1081 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1141 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1201 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1261 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1321 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1381 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     1441 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1501 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1561 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttac
     1621 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1681 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1741 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1801 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     1861 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1921 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     1981 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2041 gcgaatttta acaaaatatt aacgtttaca atttaaaagg atctaggtga agatcctttt
     2101 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2161 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2221 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2281 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2341 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2401 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2461 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2521 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2581 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2641 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2701 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2761 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2821 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2881 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2941 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3001 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3061 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3121 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacg
     3181 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3241 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     3301 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     3361 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     3421 aggatctgct catgtttgac agcttatc
//
