LOCUS       40924_18406        8248 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses E. coli codon optimized EsCas13d in the pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8248)
  AUTHORS   Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA
  TITLE     Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively
            Modulated by a WYL-Domain-Containing Accessory Protein.
  JOURNAL   Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028.
  PUBMED    29551514
REFERENCE   2  (bases 1 to 8248)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8248)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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        53..82
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             102..124
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             156..173
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        3054..3088
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119)"
     terminator      3197..3244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3281..3736
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3831..4643)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4765..5353
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5507..5524
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(5539..5678)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5764..5786
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5783..5971)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6746..6767)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6783..7862)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7863..7940)
                     /label=lacI promoter
     primer_bind     8146..8165
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttacact
       61 ttatgcttcc ggctcgtatg tttctagaga attcaaataa ttttgtttaa ctttaagaag
      121 gagatttaaa tatgaaaatc gaagaaggta aaggtcacca tcaccatcac cacatgggca
      181 aaaaaatcca tgcacgtgat ctgcgtgaac agcgtaaaac cgatcgtacc gaaaaatttg
      241 cagaccagaa caaaaaacgt gaagcagaac gtgcagttcc gaaaaaagat gcagcagtta
      301 gcgttaaaag cgttagcagc gtgagcagca aaaaagataa tgttaccaaa agcatggcaa
      361 aagcagccgg tgtgaaaagc gtttttgcag ttggtaatac cgtttacatg accagctttg
      421 gtcgtggtaa tgatgcagtt ctggaacaga aaattgttga taccagccat gaaccgctga
      481 atattgatga tccggcatat cagctgaatg tggttaccat gaatggttat agcgttaccg
      541 gtcatcgtgg tgaaaccgtt agcgcagtta ccgataatcc gctgcgtcgt tttaatggtc
      601 gtaaaaaaga cgaaccggaa cagagcgttc cgaccgatat gctgtgtctg aaaccgacac
      661 tggaaaaaaa gttttttggc aaagagttcg acgacaacat tcatatccag ctgatttata
      721 acatcctgga catcgaaaaa atcctggcag tttatagcac caatgcaatt tatgccctga
      781 ataacatgag cgcagatgaa aacattgaga acagcgactt ttttatgaaa cgcaccaccg
      841 atgaaacctt cgatgatttc gaaaaaaaga aagaaagcac caacagccgt gaaaaagcag
      901 attttgatgc cttcgaaaaa ttcatcggta attatcgcct ggcctatttt gcagatgcct
      961 tttatgtcaa caaaaagaac ccgaaaggca aagccaaaaa tgtgctgcgt gaagataaag
     1021 aactgtatag cgttctgacc ctgattggta aactgcgtca ttggtgtgtt catagcgaag
     1081 aaggtcgcgc agaattttgg ctgtataaac tggatgaact gaaggacgat ttcaaaaacg
     1141 ttctggatgt ggtttataat cgtccggtgg aagaaattaa caaccgcttt atcgaaaaca
     1201 acaaagtgaa cattcagatt ctgggcagcg tgtataaaaa caccgatatt gcagaactgg
     1261 tgcgcagcta ttatgaattt ctgatcacca aaaaatacaa aaacatgggc tttagcatca
     1321 aaaaactgcg tgaaagcatg ctggaaggta aaggttatgc cgataaagaa tatgatagcg
     1381 tgcgcaacaa actgtatcag atgaccgatt ttattctgta caccggctat atcaacgaag
     1441 atagcgatcg tgcagatgat ctggttaata ccctgcgtag cagcctgaaa gaagatgata
     1501 aaaccaccgt gtattgcaaa gaggcagatt acctttggaa aaagtatcgc gaaagcattc
     1561 gtgaagttgc cgatgcactg gatggtgaca atattaagaa actgagcaaa agcaacatcg
     1621 agatccaaga ggataaactg cgcaaatgct ttattagcta tgcagatagc gttagcgaat
     1681 ttaccaaact gatttatctg ctgacccgtt ttctgagcgg taaagaaatc aacgatctgg
     1741 tgaccacact gatcaacaaa tttgataaca ttcgcagctt tctggaaatc atggatgaat
     1801 taggtctgga tcgtaccttt accgcagaat atagcttttt tgaaggcagc accaaatatc
     1861 tggccgaact ggtggaactg aatagctttg ttaaatcctg cagcttcgat attaatgcca
     1921 aacgtaccat gtatcgtgat gccctggata ttctgggtat tgaatcagat aaaaccgaag
     1981 aggatatcga gaaaatgatc gacaatatcc tgcagattga tgccaacggt gataaaaagc
     2041 tgaaaaaaaa caatggcctg cgcaacttta ttgccagcaa tgttattgat agcaaccgct
     2101 tcaaatacct ggtgcgttat ggtaacccga agaaaattcg tgaaaccgca aaatgtaaac
     2161 cggcagttcg ttttgtgctg aatgaaattc cggatgcaca gatcgaacgt tattatgaag
     2221 catgttgtcc gaaaaatacc gcactgtgta gcgcaaataa acgtcgtgaa aaactggcag
     2281 atatgatcgc cgaaatcaaa ttcgaaaact tttccgatgc aggcaactat cagaaagcaa
     2341 atgtgaccag ccgtaccagc gaagcagaaa ttaaacgtaa aaaccaggcc attattcgcc
     2401 tgtatctgac cgttatgtat atcatgctga aaaacctggt gaatgtgaat gcccgttatg
     2461 tgattgcatt tcattgtgtt gaacgcgaca caaaactgta tgccgaaagc ggtctggaag
     2521 tgggtaatat tgaaaagaat aaaaccaacc tgaccatggc cgttatgggt gttaaactgg
     2581 aaaatggcat tatcaaaacc gagttcgata agagctttgc agaaaatgca gcaaatcgtt
     2641 atctgcgtaa tgcgcgttgg tataagctga ttctggataa tctgaagaaa agcgaacgtg
     2701 ccgttgtgaa tgaatttcgc aataccgtgt gtcatctgaa tgccattcgc aacattaaca
     2761 tcaacattaa agagatcaaa gaggtcgaga actatttcgc cctgtatcat tatctgatcc
     2821 agaaacatct ggaaaaccgc ttcgcagaca aaaaagttga acgtgatacc ggtgatttca
     2881 tcagcaaact ggaagaacat aaaacctact gcaaagactt cgtgaaagcc tattgtaccc
     2941 cgtttggtta taatctggtt cgctataaaa acctgacgat cgatggtctg ttcgacaaaa
     3001 actatccggg taaagatgat agcgacgagc agaaataata agaattcctc gagttgacag
     3061 ctagctcagt cctaggtata atgctagcag agaccatggc caccggtctc tgctaaagat
     3121 atgaggagct caaactcgag ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3181 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3241 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     3301 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3361 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3421 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3481 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3541 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3601 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3661 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3721 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3781 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     3841 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3901 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3961 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     4021 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     4081 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     4141 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     4201 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     4261 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     4321 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     4381 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     4441 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     4501 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     4561 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     4621 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     4681 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4741 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4801 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4861 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4921 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4981 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5041 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5101 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5161 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5221 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5281 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5341 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5401 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5461 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5521 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5581 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5641 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     5701 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5761 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5821 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5881 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5941 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     6001 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     6061 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     6121 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     6181 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     6241 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     6301 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     6361 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     6421 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     6481 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     6541 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     6601 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     6661 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     6721 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     6781 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6841 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6901 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6961 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     7021 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     7081 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     7141 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     7201 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     7261 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     7321 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     7381 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     7441 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     7501 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     7561 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     7621 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     7681 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     7741 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     7801 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     7861 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7921 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7981 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     8041 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     8101 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     8161 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     8221 gaggatcgag atctcgatcc cgcgaaat
//
