LOCUS       62056_306        8555 bp DNA     circular SYN 12-AUG-2020
DEFINITION  Cloning vector AGG1328:pCas13b, complete sequence.
ACCESSION   MT119959
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8555)
  AUTHORS   Tng PYL., Carabajal Paladino L, Verkuijl SAN., Purcell J, Merits A, 
            Leftwich PT, Fragkoudis R, Noad R, Alphey L.
  TITLE     Cas13b-dependent and Cas13b-independent RNA knockdown of viral 
            sequences in mosquito cells following guide RNA expression
  JOURNAL   Commun Biol 3 (1), 413 (2020)
  PUBMED    32737398
REFERENCE   2  (bases 1 to 8555)
  AUTHORS   Tng PYL., Carabajal Paladino L, Verkuijl S, Purcell J, Merits A, 
            Leftwich PT, Fragkoudis R, Noad R, Alphey L.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-FEB-2020) Arthropod Genetics Group, The Pirbright 
            Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
REFERENCE   3  (bases 1 to 8555)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol"; date: "2020"; volume: "3"; issue: "1"; pages: "413"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (26-FEB-2020) Arthropod Genetics Group, The Pirbright Institute, Ash
            Road, Pirbright, Woking, Surrey GU24 0NF, UK"
FEATURES             Location/Qualifiers
     source          1..8555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..622
                     /label=hr5 enhancer
                     /note="baculovirus early transcription enhancer"
     misc_feature    1352..4621
                     /gene="PspCas13b-T2A-ZsYellow"
                     /label=PspCas13b
                     /note="PspCas13b"
     misc_feature    4628..4669
                     /gene="PspCas13b-T2A-ZsYellow"
                     /label=super-PK1 NES
                     /note="super-PK1 NES"
     CDS             4679..4705
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             4706..4732
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             4733..4759
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     misc_feature    4760..4788
                     /gene="PspCas13b-T2A-ZsYellow"
                     /label=thoseaasigna virus 2A
                     /note="thoseaasigna virus 2A"
     misc_feature    4789..4813
                     /gene="PspCas13b-T2A-ZsYellow"
                     /label=thoseaasigna virus 2A
                     /note="thoseaasigna virus 2A"
     misc_feature    4814..4837
                     /gene="PspCas13b-T2A-ZsYellow"
                     /label=nuclear localization signal
                     /note="nuclear localization signal"
     CDS             4844..5536
                     /label=ZsYellow1
                     /note="Zoanthus yellow fluorescent protein"
     CDS             5546..5566
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     polyA_signal    complement(5623..5744)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(6151..6739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6913..7770)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7771..7875)
                     /label=AmpR promoter
     rep_origin      7902..8357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     polyA_signal    8488..8536
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     misc_feature    8550..8555
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"
ORIGIN
        1 cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc
       61 aagtgcaggt gccagaacat ttctctatcg ataggtaccg agctcttacg cgtgctagag
      121 ctttacgagt agaattctac gcgtaaaaca caatcaagta tgagtcataa tctgatgtca
      181 tgttttgtac acggctcata accgaactgg ctttacgagt agaattctac ttgtaatgca
      241 cgatcagtgg atgatgtcat ttgtttttca aatcgagatg atgtcatgtt ttgcacacgg
      301 ctcataaact cgctttacga gtagaattct acgtgtaacg cacgatcgat tgatgagtca
      361 tttgttttgc aatatgatat catacaatat gactcatttg tttttcaaaa ccgaacttga
      421 tttacgggta gaattctact tgtaaagcac aatcaaaaag atgatgtcat ttgtttttca
      481 aaactgaact cgctttacga gtagaattct acgtgtaaaa cacaatcaag aaatgatgtc
      541 atttgttata aaaataaaag ctgatgtcat gttttgcaca tggctcataa ctaaactcgc
      601 tttacgggta gaattctacg cgtaaaacat gattgataat taaataattc atttgcaagc
      661 tatacgttaa atcaaacgga cgctcgaggt tgcacaacac tattatcgat ttgcagttcg
      721 ggacataaat gtttaaatat atcgatgtct ttgtgatgcg cgcgacattt ttgtaggtta
      781 ttgataaaat gaacggatac gttgcccgac attatcatta aatccttggc gtagaatttg
      841 tcgggtccat tgtccgtgtg cgctagtagc atgcccgtaa cggacctcgt acttttggct
      901 tcaaaggttt tgcgcacaga caaaatgtgc cacacttgca gctctgcatg tgtgcgcgtt
      961 accacaaatc ccaacggcgc agtgtacttg ttgtatgcaa ataaatctcg ataaaggcgc
     1021 ggcgcgcgaa tgcagctgat cacgtacgct cctcgtgttc cgttcaagga cggtgttatc
     1081 gacctcagat taatgtttat cggccgactg ttttcgtatc cgctcaccaa acgcgttttt
     1141 gcattaacat tgtatgtcgg cggatgttct atatctaatt tgaataaata aacgataacc
     1201 gcgttggttt tagagggcat aataaaagaa atattgttat cgtgttcgcc attagggcag
     1261 tataaattga cgttcatgtt ggatattgtt tcagttgcaa gttgacactg gcggcgacaa
     1321 gaggcgcgcc taccgcggct cgaacgccac catgaacatc ccggccctgg tggaaaacca
     1381 gaagaagtac ttcggaacct actcggtgat ggccatgctg aacgcccaga ccgtgctgga
     1441 tcacatccag aaggtggccg atatcgaagg agaacagaac gaaaacaacg aaaacctgtg
     1501 gttccacccg gtgatgtcgc acctgtacaa cgccaagaac ggatacgata agcagccgga
     1561 aaagaccatg ttcatcatcg aacgtctcca gagttacttc ccgttcctga aaatcatggc
     1621 cgaaaaccag cgtgaatact cgaacggaaa gtacaagcag aaccgtgtgg aagtgaactc
     1681 gaacgatatc tttgaagtgc tgaagcgtgc cttcggagtg ctgaagatgt accgtgatct
     1741 gaccaaccac tacaagacct acgaagaaaa gctgaacgat ggatgcgagt tcctgacctc
     1801 gaccgaacag ccgctgtcgg gaatgatcaa caactactac accgtggccc tgcgtaacat
     1861 gaacgaacgt tacggataca agaccgagga cctggcattc atccaggata agcgtttcaa
     1921 gttcgtgaag gatgcctacg gaaagaagaa gtcgcaagtg aacaccggat tcttcctgtc
     1981 gttgcaagac tacaacggag atacccagaa gaagctgcac ctgtcgggag tgggaatcgc
     2041 cctgctgatc tgcctgttcc tggataagca gtacatcaac atcttcctgt cgcgtctgcc
     2101 gatcttctcg tcgtacaacg cccagtcgga agaacgtcgt atcatcatcc gttcgttcgg
     2161 aatcaactcg atcaagctgc cgaaggatcg tatccactcg gaaaagtcga acaagtcggt
     2221 ggcaatggat atgctgaacg aagtgaagcg ttgcccggat gaactgttca ccaccctgtc
     2281 ggccgaaaag cagtcgcgtt tccgtatcat ctcggatgat cacaacgaag tgctgatgaa
     2341 gcgttcgtcg gatcgtttcg tgccgctgct gctccaatac atcgattacg gaaagctgtt
     2401 cgatcacatc cgtttccacg tgaacatggg aaagctgcgt tacctgctga aggccgataa
     2461 gacctgcatc gatggacaga cccgtgtgcg tgtgatcgaa cagccgctga acggattcgg
     2521 acgcctggaa gaagccgaaa ccatgcgcaa gcaggaaaac ggaaccttcg gaaacagtgg
     2581 aatccgtatc cgtgatttcg agaacatgaa gcgtgatgat gccaacccgg ccaactaccc
     2641 gtacatcgtg gatacctaca cccactacat cctggaaaac aacaaggtgg aaatgttcat
     2701 caacgataag gaagattcgg ccccgctgct gccggtgatc gaagatgatc gttacgtggt
     2761 gaagaccatt ccgtcgtgcc gtatgtcgac cctggaaatc ccggcaatgg ccttccacat
     2821 gttcctgttc ggatcgaaga agaccgaaaa gctgatcgtg gatgtgcaca accgttacaa
     2881 gcgtctgttc caagccatgc agaaggaaga agtgaccgcc gaaaacatcg cctcgttcgg
     2941 aatcgccgaa tcggatctgc cgcagaagat cctggatctg atctccggaa acgcccacgg
     3001 aaaggatgtg gatgccttca tccgtctgac cgtggatgat atgctgaccg ataccgaacg
     3061 tcgtatcaag cgtttcaagg atgatcgcaa gtcgatccgt tcggccgata acaagatggg
     3121 aaagcgtgga ttcaagcaga tctcgaccgg aaagctggcc gatttcctgg ccaaagatat
     3181 cgtgctgttc caaccgtcgg tgaacgatgg agaaaacaag atcaccggac tgaactaccg
     3241 tatcatgcag tcggccatcg ccgtgtacga ttcgggagat gattacgaag ccaagcagca
     3301 gttcaagctg atgtttgaaa aggcccgtct gatcggaaag ggaaccaccg aaccgcaccc
     3361 gttcctgtac aaggtgttcg cccgttcgat cccggccaac gccgtggaat tttacgaacg
     3421 ttacctgatc gaacgcaagt tctacctgac cggactgtcg aacgaaatca agaagggaaa
     3481 ccgtgtggat gtgccgttca tccgtcgtga tcagaacaag tggaagaccc cggccatgaa
     3541 gaccctggga cgtatctact cggaagatct gccggtggaa ctgccgcgtc agatgttcga
     3601 taacgaaatc aagtcgcacc tgaagtcgct gccgcagatg gaaggaatcg atttcaacaa
     3661 cgccaacgtg acctacctga tcgccgaata catgaagcgt gtgctggatg atgatttcca
     3721 gaccttctac cagtggaacc gcaactaccg ttacatggat atgctgaagg gagaatacga
     3781 tcgcaaggga tcgctccagc actgcttcac ctcggtggaa gaacgtgaag gactgtggaa
     3841 ggaacgtgcc tcgcgtaccg aacgttaccg caagcaggcc tcgaacaaga tccgtagtaa
     3901 ccgtcagatg cgtaacgcct cgtcggaaga aatcgaaacc atcctggata agcgtctgtc
     3961 gaactcgcgt aacgaatacc agaagtcgga aaaggtcatc cgtcgttacc gtgtgcaaga
     4021 tgccctgctg ttcctgctgg ccaagaagac cctgaccgaa ctggccgatt tcgatggaga
     4081 acgtttcaag ctgaaggaaa tcatgccgga tgccgaaaag ggaatcctgt cggaaatcat
     4141 gccgatgtcg ttcacctttg aaaagggagg aaagaagtac accatcacct cggaaggaat
     4201 gaagctgaag aactacggag atttcttcgt gctggcctcg gataagcgta tcggaaacct
     4261 gctggaattg gtcggatcgg atatcgtgtc gaaggaagat atcatggaag aatttaacaa
     4321 atacgatcag tgccgtccgg aaatctcgtc gatcgtgttc aacctggaaa agtgggcctt
     4381 cgatacctac ccggaactgt cggcccgtgt ggatcgtgaa gaaaaggtcg atttcaagtc
     4441 gatcctgaag atcctgctga acaacaagaa catcaacaag gaacagtcgg atatcctgcg
     4501 caagatccgt aacgccttcg atcacaacaa ctacccggat aagggagtgg tggaaatcaa
     4561 ggccctgccg gaaatcgcca tgtcgatcaa gaaggccttc ggagaatacg ccatcatgaa
     4621 gggatcgaac atcaacgaac tggccctgaa gttcgccgga ctggatctgg gatcgtcgta
     4681 tccgtacgac gtgccggatt atgcctaccc ctacgatgtg ccagattacg cctacccgta
     4741 cgacgtcccg gattatgccg agggcagagg aagtcttcta acatgcggtg acgtggagga
     4801 gaatcccggc cctaccccac ccaagaagaa gcgcaaagct agcatggccc acagcaagca
     4861 cggcctgaag gaggagatga ccatgaagta ccacatggag ggctgcgtga acggccacaa
     4921 gttcgtgatc accggcgagg gcatcggcta ccccttcaag ggcaagcaga ccatcaacct
     4981 gtgcgtgatc gagggcggcc ccctgccctt cagcgaggac atcctgagcg ccggcttcaa
     5041 gtacggcgac cggatcttca ccgagtaccc ccaggacatc gtggactact tcaagaacag
     5101 ctgccccgcc ggctacacct ggggccggag cttcctgttc gaggacggcg ccgtgtgcat
     5161 ctgtaacgtg gacatcaccg tgagcgtgaa ggagaactgc atctaccaca agagcatctt
     5221 caacggcgtg aacttccccg ccgacggccc cgtgatgaag aagatgacca ccaactggga
     5281 ggccagctgc gagaagatca tgcccgtgcc taagcagggc atcctgaagg gcgatgtcag
     5341 catgtacctg ctgctgaagg acggcggccg gtaccggtgc cagttcgaca ccgtgtacaa
     5401 ggccaagagc gtgcccagca agatgcccga gtggcacttc atccagcaca agctgctgcg
     5461 ggaggaccgg agcgacgcca agaaccagaa gtggcagctg accgagcacg ccatcgcctt
     5521 ccccagcgcc ctggccagat ctcgacccaa gaaaaagcgg aaggtggagg acccgtaact
     5581 aaataatgat ccgggcccat ctagagcttc gagcagacat gataagatac attgatgagt
     5641 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5701 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     5761 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     5821 tctacaaatg tggtaaaatg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     5881 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     5941 caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg
     6001 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6061 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6121 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6181 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6241 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6301 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6361 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6421 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6481 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6541 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6601 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6661 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6721 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6781 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6841 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6901 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6961 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7021 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7081 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7141 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7201 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7261 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7321 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7381 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7441 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7501 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7561 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7621 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7681 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7741 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7801 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7861 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     7921 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     7981 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     8041 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     8101 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     8161 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     8221 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     8281 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     8341 attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     8401 ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa
     8461 ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt
     8521 gttggttttt tgtgtgaatc gatagtacta acata
//
