LOCUS       sequence-258107-        4410 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 4410)
  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 4410)
  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..4410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..44
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             243..254
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             261..974
                     /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      986..1057
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1073..1100
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1192..1209)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1362..1448
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1540..1567
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1586..1677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1678..1746,1747..2538)
                     /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(1896..1915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2580..3035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2667..2686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2877..2898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3146..3734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3635..3654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3888..3905
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3920..4060
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3986..4005)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4146..4168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4306..4325
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgatgcatt tgacagctag ctcagtccta ggtataatgc tagcgctagc gaattcgagc
       61 tcggtaccaa gacgtcaact cgaccctcga gacggaaagt gaaacgtgat ttcatgcgtc
      121 attttgaaca ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca tttaatttat
      181 gaatgttttc ttaacatcgc ggcaactcaa gaaacggcag gttcggatct tagctactag
      241 agaaagagga gaaatactag atgcgtaaag gcgaagagct gttcactggt gtcgtcccta
      301 ttctggtgga actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg
      361 aaggtgacgc aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc
      421 cggttccttg gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt
      481 atccggacca tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc
      541 aggaacgcac gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat
      601 ttgaaggcga taccctggta aaccgcattg agctgaaagg cattgacttt aaagaggacg
      661 gcaatatcct gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg
      721 ccgataaaca aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg
      781 gcagcgtgca gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc
      841 tgctgccaga caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga
      901 aacgcgatca tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg
      961 atgaactgta caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1021 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     1081 ttcgggtggg cctttctgcg tttatacgtc tctaacggat atagaccacc ccaatatcga
     1141 aggggactaa aacaagcttg gctgttttgg cggatgagag aagattttca gcctgataca
     1201 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
     1261 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
     1321 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
     1381 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     1441 ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg
     1501 caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg
     1561 gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc aaatatgtat
     1621 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1681 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1741 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1801 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1861 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1921 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1981 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2041 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2101 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2161 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2221 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2281 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2341 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2401 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2461 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2521 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2581 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2641 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2701 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2761 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     2821 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2881 actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata
     2941 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3001 cgcgaatttt aacaaaatat taacgtttac aatttaaaag gatctaggtg aagatccttt
     3061 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3661 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3721 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3781 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3841 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3901 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3961 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4021 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4081 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4141 ggtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4201 gaaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     4261 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     4321 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     4381 gaggatctgc tcatgtttga cagcttatca
//
