LOCUS       sequence-325295-        3420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of single-spacer CRISPR array with 
            non-targeting spacer for LsCas13a in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3420)
  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 3420)
  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-NT
FEATURES             Location/Qualifiers
     source          1..3420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        165..199
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(357..374)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      527..613
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      705..732
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        751..842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(843..911,912..1703)
                     /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(1061..1080)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1832..1851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2042..2063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      2311..2899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2800..2819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3053..3070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3085..3225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3151..3170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3311..3333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
       61 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      121 gcgtagagga tctgctcatg tttgacagct tatcatcgat gcatttgaca gctagctcag
      181 tcctaggtat aatgctagcg ctagcgaatt cgagctcggt accaagacgt caactcgacc
      241 ctgatataga ccaccccaat atcgaagggg actaaaacaa cggatataga ccaccccaat
      301 atcgaagggg actaaaacaa gcttggctgt tttggcggat gagagaagat tttcagcctg
      361 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
      421 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
      481 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
      541 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
      601 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
      661 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
      721 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
      781 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      841 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      901 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      961 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1021 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1081 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1141 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1201 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1261 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1321 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1381 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1441 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1501 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1561 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1621 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1681 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1741 atttacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1801 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     1981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2041 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     2101 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2161 tttaacgcga attttaacaa aatattaacg tttacaattt aaaaggatct aggtgaagat
     2221 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2281 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2341 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2401 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2461 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2521 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2581 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2641 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2701 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2761 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2821 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2881 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2941 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3001 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3061 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3121 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3181 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3241 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3301 gtgacggtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3361 aggcagaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
//
