LOCUS       62056_316        5623 bp DNA     circular SYN 12-AUG-2020
DEFINITION  Cloning vector AGG1521:pCHIKVRep1, complete sequence.
ACCESSION   MT119957
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5623)
  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 5623)
  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 5623)
  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..5623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(227..1033)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1525..1541
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2526..3242
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3424..3936
                     /codon_start=1
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    4533..4654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4679..4695)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4703..4719)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4727..4757)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4772..4793)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(join(5081..5623,1..46))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
       61 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
      121 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
      181 aagcccaatc tgaataatgt tacaaccaat taaccaattc tgattagaaa aactcatcga
      241 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
      301 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
      361 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
      421 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
      481 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
      541 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
      601 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
      661 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
      721 atgctgtttt tccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
      781 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
      841 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
      901 gcttcccata caagcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
      961 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgac gtttcccgtt
     1021 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     1081 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acgggccaga
     1141 gctgcatcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     1201 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     1261 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     1321 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     1381 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     1441 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     1501 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattctat ctttacatgt
     1561 agcttgtgca ttgaatccaa ttataatttg ccttggcacc agctgagcca gacaagaaag
     1621 aaagcttccc agaagtatat cgatttagaa gggttgacgt cacttgctga ctgcactaat
     1681 acagcaaatg atgcaattag aatgattcaa gtgaaattcc caaattactg atttttctct
     1741 ggatttggtt atcagattac attcgaagct aagattagct accgaaattg tcgatcaaat
     1801 caggaaatcc tttctctatc gaaaaaggca ttcgcacatc ttcctctcta tgccatatac
     1861 acgaagggta ggtacattga cgtctttgcc agaagttgaa ctgcatcgtt caaggtacag
     1921 aatgaacgac taacagacac aagcacgttt tgctgtccat tcagacacag ggatggtacc
     1981 catattcgat cgatatagag ccatccaacc gaacagaggt atatgtatga atgtattgct
     2041 gaaattttct agaagtacaa ccaccactac gacagtgtct ataaaacgcc cctgcaaagg
     2101 cgaaaccagc tcatggctgc gtgagacaca cgtagcctac cagtttctta ctgctctact
     2161 ctgcaaagca agagattaat aacccatcat ggatcctgtg tacgtggaca tagacgctga
     2221 cagcgccttt ttgaaggccc tgcaacgtgc gtaccccatg tttgaggtgg aaccaaggca
     2281 ggtcacaccg aatgaccatg ctaatgctag agcgttctcg catctagcta taaaactaat
     2341 agagcaggaa attgaccccg actcaaccat cctggatatc ggcagtgcgc cagcaaggag
     2401 gatgatgtcg gacaggaagt accctggcgg taagttgatc aaaggaaacg caaagttttc
     2461 aagaaaaaac aaaactaatt tgatttataa cacctttaga aagcggggca ctcgagtatg
     2521 ggcccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2581 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2641 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2701 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2761 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2821 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2881 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2941 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     3001 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3061 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3121 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     3181 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3241 agagatctta atccggttaa ttaattaaca tggccacctt tgcaagctcc agatccaact
     3301 tcgagaagct cagaggaccc gtcataactt tgtacggcgg tcctaaatag gtacgcacta
     3361 cagctaccta ttttgcagaa gccgacagca agtatctaaa cactaatcag ctacagcggc
     3421 cgcatggtgt tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac
     3481 ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc
     3541 gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat
     3601 gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt
     3661 aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg
     3721 gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc
     3781 gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt
     3841 atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga
     3901 gtgaccggct ggcggctgtg cgaacgcatt ctggcgtaaa gatcttaagt cgactaatta
     3961 attagcctaa aagcagccga actcactttg agaagtaggc atagcatacc gaactcttcc
     4021 acgattctcc gaacccacag ggacgtagga gatgttattt tgtttttaat atttcaaaaa
     4081 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4141 agggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccga aggaggacgc
     4201 acgtccactc ggatggctaa gggagcctgc attcgcagaa gcccagcaca ctggcggccg
     4261 ttactagggc cgcgcccttc ccaacagttg cgcagcctga atggcgaatg gagatccaat
     4321 ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattcaca
     4381 gtcccaaggc tcatttcagg cccctcagtc ctcacagtct gttcatgatc ataatcagcc
     4441 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     4501 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4621 gttgtggttt gtccaaactc atcaatgtat cttacccggg aagcttggcg taatcatggt
     4681 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4741 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4921 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4981 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5041 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5101 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5161 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5221 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5281 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5341 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5401 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5461 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5521 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5581 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg ttt
//
