LOCUS       sequence-252345-        4411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for generating CRISPR arrays for LsCas13a. Contains
            a GFP-dropout cassette and a direct repeat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4411)
  AUTHORS   Liao C, Ttofali F, Slotkowski RA, Denny SR, Cecil TD, Leenay RT, 
            Keung AJ, Beisel CL
  TITLE     Modular one-pot assembly of CRISPR arrays enables library generation
            and reveals factors influencing crRNA biogenesis.
  JOURNAL   Nat Commun. 2019 Jul 3;10(1):2948. doi: 10.1038/s41467-019-10747-3.
  PUBMED    31270316
REFERENCE   2  (bases 1 to 4411)
  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: "2019-07-3"; volume: "10(1):2948. doi"; pages: " 
            10.1038/s41467-019-10747-3"
COMMENT     Sequence Label: pLsCas13aGG
FEATURES             Location/Qualifiers
     source          1..4411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     166..185
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        280..314
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             513..524
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             531..1244
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1256..1327
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1343..1370
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1462..1479)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1632..1718
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1810..1837
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1856..1947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1948..2016,2017..2808)
                     /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(2166..2185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2850..3305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2937..2956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      3416..4004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3905..3924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4158..4175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4190..4330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4256..4275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
       61 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
      121 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      181 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      241 gaggatctgc tcatgtttga cagcttatca tcgatgcatt tgacagctag ctcagtccta
      301 ggtataatgc tagcgctagc gaattcgagc tcggtaccaa gacgtcaact cgaccctcga
      361 gacggaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat cttatttaat
      421 aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc ggcaactcaa
      481 gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag atgcgtaaag
      541 gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg
      601 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
      661 tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc
      721 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
      781 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
      841 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
      901 agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca
      961 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
     1021 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
     1081 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
     1141 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
     1201 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat
     1261 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1321 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatacgtc
     1381 tctaacggat atagaccacc ccaatatcga aggggactaa aacaagcttg gctgttttgg
     1441 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     1501 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     1561 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     1621 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1681 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     1741 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     1801 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     1861 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1921 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1981 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2041 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2101 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2161 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     2221 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2281 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2341 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2401 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2461 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2521 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2581 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2641 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2701 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2761 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2821 gtttactcat atatacttta gattgattta cgcgccctgt agcggcgcat taagcgcggc
     2881 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2941 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3001 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3061 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3121 gacgttggag tccacgttct ttaatagtgg actcttgttc caaacttgaa caacactcaa
     3181 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3241 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3301 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3361 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3421 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3481 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3541 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3601 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3661 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3721 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3781 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3841 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3901 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3961 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4021 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4081 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4141 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4201 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4261 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4321 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4381 cccggcatcc gcttacagac aagctgtgac g
//
