LOCUS       Exported                2914 bp ds-DNA     circular SYN 10-DEC-2024
DEFINITION  Backbone for cloning LwCas13a guides under U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pC016 - LwCas13a guide expression backbone with U6 promoter
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2914)
  AUTHORS   Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, 
            Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, 
            Voytas DF, Ting AY, Zhang F
  TITLE     RNA targeting with CRISPR-Cas13.
  JOURNAL   Nature. 2017 Oct 4. doi: 10.1038/nature24049.
  PUBMED    28976959
REFERENCE   2  (bases 1 to 2914)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jan 28, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2914
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      138..726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     627..646
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    789..1037
                     /label=hU6
     promoter        789..1029
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     789..809
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     960..979
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1008..1037)
                     /label=AA_060R
     primer_bind     1018..1079
                     /label=AA_599
     primer_bind     1018..1037
                     /label=AA_059F
     misc_feature    1038..1060
                     /label=AUG_i
     misc_feature    1038..1060
                     /label=23nt
     misc_feature    1038..1059
                     /label=30nt target
     CDS             complement(1038..1047)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVS"
     primer_bind     complement(1061..1128)
                     /label=AA_058R
     misc_feature    1061..1096
                     /label=DR
     primer_bind     complement(1097..1128)
                     /label=AA_062
     misc_feature    1097..1104
                     /label=U6 term
     primer_bind     1105..1134
                     /label=AA_061F
     rep_origin      1179..1634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1266..1285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1476..1497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1651..1670)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1770..1792
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1830..1848)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1916..2020
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2021..2881
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2239..2258)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
       61 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      121 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      181 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      241 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      301 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      361 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      421 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      481 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      541 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      601 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      661 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      721 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      781 cagctagcga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      841 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      901 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      961 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1021 ggaaaggacg aaacacctgc tcaccatggt agcttaagtt gttgtggaag gtccagtttt
     1081 gaggggctat tacaactttt ttttgaattc tgatgcggta ttttctcctt acgcatctgt
     1141 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     1201 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     1261 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1321 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1381 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1441 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1501 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     1561 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1621 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1681 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1741 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     1801 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     1861 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     1921 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1981 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2041 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2101 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2161 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2221 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     2281 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2341 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2401 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2461 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2521 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2581 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2641 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2701 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     2761 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2821 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2881 actgtcagac caagtttact catatatact ttag
//
