LOCUS       Exported                6158 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid to carry out IVT of RfxCas13d-NLS (human codon-optimized).
ACCESSION   .
VERSION     .
KEYWORDS    pT3TS-RfxCas13d-NLS-HA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6158)
  AUTHORS   Kushawah G, Hernandez-Huertas L, Abugattas-Nunez del Prado J, 
            Martinez-Morales JR, DeVore ML, Hassan H, Moreno-Sanchez I, 
            Tomas-Gallardo L, Diaz-Moscoso A, Monges DE, Guelfo JR, Theune WC, 
            Brannan EO, Wang W, Corbin TJ, Moran AM, Sanchez Alvarado A, 
            Malaga-Trillo E, Takacs CM, Bazzini AA, Moreno-Mateos MA
  TITLE     CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos
  JOURNAL   Developmental Cell. 2020.
REFERENCE   2  (bases 1 to 6158)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6158
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      102..111
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             117..137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             147..3044
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3054..3074
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3084..3110
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     3'UTR           3135..3260
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(3346..3365)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3347..3365)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3372..3389)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3372..3388)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3381..3403)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3530..3985)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3667..3688)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3879..3898
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4012..4116
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4117..4977
                     /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(4335..4354)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5148..5736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5637..5656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5890..5907
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6024..6045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6060..6090
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6098..6114
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     6103..6125
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6122..6138
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6122..6138
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg gcgagcccca
      121 agaagaagag aaaggtggag gccagcatcg aaaaaaaaaa gtccttcgcc aagggcatgg
      181 gcgtgaagtc cacactcgtg tccggctcca aagtgtacat gacaaccttc gccgaaggca
      241 gcgacgccag gctggaaaag atcgtggagg gcgacagcat caggagcgtg aatgagggcg
      301 aggccttcag cgctgaaatg gccgataaaa acgccggcta taagatcggc aacgccaaat
      361 tcagccatcc taagggctac gccgtggtgg ctaacaaccc tctgtataca ggacccgtcc
      421 agcaggatat gctcggcctg aaggaaactc tggaaaagag gtacttcggc gagagcgctg
      481 atggcaatga caatatttgt atccaggtga tccataacat cctggacatt gaaaaaatcc
      541 tcgccgaata cattaccaac gccgcctacg ccgtcaacaa tatctccggc ctggataagg
      601 acattattgg attcggcaag ttctccacag tgtataccta cgacgaattc aaagaccccg
      661 agcaccatag ggccgctttc aacaataacg ataagctcat caacgccatc aaggcccagt
      721 atgacgagtt cgacaacttc ctcgataacc ccagactcgg ctatttcggc caggcctttt
      781 tcagcaagga gggcagaaat tacatcatca attacggcaa cgaatgctat gacattctgg
      841 ccctcctgag cggactgagg cactgggtgg tccataacaa cgaagaagag tccaggatct
      901 ccaggacctg gctctacaac ctcgataaga acctcgacaa cgaatacatc tccaccctca
      961 actacctcta cgacaggatc accaatgagc tgaccaactc cttctccaag aactccgccg
     1021 ccaacgtgaa ctatattgcc gaaactctgg gaatcaaccc tgccgaattc gccgaacaat
     1081 atttcagatt cagcattatg aaagagcaga aaaacctcgg attcaatatc accaagctca
     1141 gggaagtgat gctggacagg aaggatatgt ccgagatcag gaaaaatcat aaggtgttcg
     1201 actccatcag gaccaaggtc tacaccatga tggactttgt gatttatagg tattacatcg
     1261 aagaggatgc caaggtggct gccgccaata agtccctccc cgataatgag aagtccctga
     1321 gcgagaagga tatctttgtg attaacctga ggggctcctt caacgacgac cagaaggatg
     1381 ccctctacta cgatgaagct aatagaattt ggagaaagct cgaaaatatc atgcacaaca
     1441 tcaaggaatt taggggaaac aagacaagag agtataagaa gaaggacgcc cctagactgc
     1501 ccagaatcct gcccgctggc cgtgatgttt ccgccttcag caaactcatg tatgccctga
     1561 ccatgttcct ggatggcaag gagatcaacg acctcctgac caccctgatt aataaattcg
     1621 ataacatcca gagcttcctg aaggtgatgc ctctcatcgg agtcaacgct aagttcgtgg
     1681 aggaatacgc ctttttcaaa gactccgcca agatcgccga tgagctgagg ctgatcaagt
     1741 ccttcgctag aatgggagaa cctattgccg atgccaggag ggccatgtat atcgacgcca
     1801 tccgtatttt aggaaccaac ctgtcctatg atgagctcaa ggccctcgcc gacacctttt
     1861 ccctggacga gaacggaaac aagctcaaga aaggcaagca cggcatgaga aatttcatta
     1921 ttaataacgt gatcagcaat aaaaggttcc actacctgat cagatacggt gatcctgccc
     1981 acctccatga gatcgccaaa aacgaggccg tggtgaagtt cgtgctcggc aggatcgctg
     2041 acatccagaa aaaacagggc cagaacggca agaaccagat cgacaggtac tacgaaactt
     2101 gtatcggaaa ggataagggc aagagcgtga gcgaaaaggt ggacgctctc acaaagatca
     2161 tcaccggaat gaactacgac caattcgaca agaaaaggag cgtcattgag gacaccggca
     2221 gggaaaacgc cgagagggag aagtttaaaa agatcatcag cctgtacctc accgtgatct
     2281 accacatcct caagaatatt gtcaatatca acgccaggta cgtcatcgga ttccattgcg
     2341 tcgagcgtga tgctcaactg tacaaggaga aaggctacga catcaatctc aagaaactgg
     2401 aagagaaggg attcagctcc gtcaccaagc tctgcgctgg cattgatgaa actgcccccg
     2461 ataagagaaa ggacgtggaa aaggagatgg ctgaaagagc caaggagagc attgacagcc
     2521 tcgagagcgc caaccccaag ctgtatgcca attacatcaa atacagcgac gagaagaaag
     2581 ccgaggagtt caccaggcag attaacaggg agaaggccaa aaccgccctg aacgcctacc
     2641 tgaggaacac caagtggaat gtgatcatca gggaggacct cctgagaatt gacaacaaga
     2701 catgtaccct gttcagaaac aaggccgtcc acctggaagt ggccaggtat gtccacgcct
     2761 atatcaacga cattgccgag gtcaattcct acttccaact gtaccattac atcatgcaga
     2821 gaattatcat gaatgagagg tacgagaaaa gcagcggaaa ggtgtccgag tacttcgacg
     2881 ctgtgaatga cgagaagaag tacaacgata ggctcctgaa actgctgtgt gtgcctttcg
     2941 gctactgtat ccccaggttt aagaacctga gcatcgaggc cctgttcgat aggaacgagg
     3001 ccgccaagtt cgacaaggag aaaaagaagg tgtccggcaa ttccggatcc ggacctaaga
     3061 aaaagaggaa ggtggcggcc gcttacccat acgatgttcc agattacgct taaccgcgga
     3121 tctggttacc actaaaccag cctcaagaac acccgaatgg agtctctaag ctacataata
     3181 ccaacttaca ctttacaaaa tgttgtcccc caaaatgtag ccattcgtat ctgctcctaa
     3241 taaaaagaaa gtttcttcac attctaaaaa aaaaaaaaaa aaaaaaaaaa aaaacccccc
     3301 cccccccctg cagggccaag tcggcctcta gaggtaccca attcgcccta tagtgagtcg
     3361 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     3421 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     3481 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca aattgtaagc
     3541 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3601 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3661 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3721 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3781 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3841 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3901 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3961 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4021 cctatttgtt tatttttcta aatacattca aatatgtatc tgctcatgag acaataaccc
     4081 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4141 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4201 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4261 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4321 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4381 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4441 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4501 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4561 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4621 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4681 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4741 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4801 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4861 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4921 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4981 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5041 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5101 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5161 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5221 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5281 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5341 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5401 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5461 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5521 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5581 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5641 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5701 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5761 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5821 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5881 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5941 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6001 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     6061 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6121 acaggaaaca gctatgacca tgattacgcc aagctcga
//
