LOCUS       40924_18411        8149 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses E. coli codon optimized RspCas13d in the pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8149)
  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 8149)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8149)
  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..8149
                     /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        2955..2989
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119)"
     terminator      3098..3145
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3182..3637
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3732..4544)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4666..5254
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5408..5425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(5440..5579)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5665..5687
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5684..5872)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6647..6668)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6684..7763)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7764..7841)
                     /label=lacI promoter
     primer_bind     8047..8066
                     /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 cacatggcca
      181 aaaagaacaa aatgaaaccg cgtgaactgc gtgaagcaca gaaaaaagca cgtcagctga
      241 aagcagcaga aattaacaat aatgcagcac cggcaattgc agcaatgcct gcagcagaag
      301 ttattgcacc ggttgcagaa aaaaagaaaa gcagcgttaa agcagccggt atgaaaagca
      361 ttctggtgag caaaaacaaa atgtatatca ccagctttgg caaaggtaat agcgcagttc
      421 tggaatatga agtggataac aacgattaca atcagaccca gctgagcagc aaaggtagca
      481 gcaatattga gctgcgtggt gttaatgaag tgaacattac ctttagcagc aaacacggtt
      541 ttgaaagcgg tgttgaaatc aataccagca atccgacaca tcgtagcggt gaaagcagtc
      601 cggttcgtgg tgatatgctg ggcctgaaaa gcgaactgga aaaacgcttt tttggcaaaa
      661 ccttcgatga caacattcat atccagctga tctataacat cctggacatc gaaaaaattc
      721 tggccgttta tgtgaccaac attgtgtatg cactgaataa catgctgagc attaaagata
      781 gcgagagcta tgatgatttc atgggttatc tgagcgcacg caatacctat gaagttttta
      841 cccatccgga taaaagcaac ctgagcgata aagcaaaagg caacatcaaa aaatccttta
      901 gcaccttcaa cgacctgctg aaaaccaaac gtctgggtta ttttggtctg gaagaaccga
      961 aaacaaaaga tacccgtgtt agccaggcat acaaaaaacg tgtttatcac atgctggcaa
     1021 ttgtgggtca gattcgtcag agcgtgtttc atgataaaag tagcaaactg gatgaggacc
     1081 tgtatagctt tatcgatatc atcgattccg aatatcgtga aaccctggat tatctggttg
     1141 atgaacgttt tgacagcatc aacaaaggtt ttatccaggg caacaaagtg aatattagcc
     1201 tgctgatcga tatgatgaaa ggctatgaag ccgatgatat tatccgcctg tattatgatt
     1261 ttatcgtgct gaaaagccag aagaatctgg gctttagtat caaaaaactg cgcgagaaaa
     1321 tgctggatga atatggcttt cgcttcaaag ataaacagta tgatagcgtg cgtagcaaga
     1381 tgtataaact gatggatttt ctgctgttct gcaactatta tcgcaatgat gttgttgccg
     1441 gtgaagcact ggttcgtaaa ctgcgtttta gcatgaccga tgatgaaaaa gaaggtattt
     1501 acgcagacga agcaagcaaa ctgtggggta aatttcgtaa cgattttgag aacattgccg
     1561 accatatgaa cggtgatgtt attaaagaac tgggcaaagc cgatatggat ttcgatgaaa
     1621 aaatactgga cagcgagaaa aaaaacgcaa gcgatctgct gtatttcagc aagatgattt
     1681 atatgctgac ctatttcctg gatggcaaag aaattaatga tctgctgacc acgctgatta
     1741 gcaaatttga taacatcaaa gaatttctga aaatcatgaa aagctcagcc gttgatgttg
     1801 aatgtgaact gaccgcaggt tataaactgt ttaatgatag ccagcgcatt accaacgaac
     1861 tgttcattgt taaaaacatt gcgagcatgc gtaaaccggc aagcagcgca aaactgacca
     1921 tgtttcgtga tgcactgacc attctgggta ttgatgataa cattaccgat gatcgcatta
     1981 gcgaaatcct gaagctgaaa gaaaaaggta aaggtattca tggcctgcgc aactttatta
     2041 ccaataacgt gattgaaagc tcccgctttg tgtacctgat caaatatgca aatgcccaga
     2101 aaattcgcaa agtggcgaaa aatgaaaagg tggtgatgtt tgtgttaggt ggtattccgg
     2161 atacacagat tgaacgctat tacaaaagct gcgttgaatt tccggatatg aacagcagcc
     2221 tggaagttaa acgtagtgaa ctggcacgta tgatcaaaaa catcagcttc gacgatttca
     2281 agaacgttaa acagcaggca aaaggtcgtg aaaatgttgc aaaagaacgt gccaaagcag
     2341 tgattggtct gtatctgacc gttatgtatc tgctggttaa aaatctggtt aacgtgaatg
     2401 cccgttatgt gattgcaatt cattgtctgg aacgtgattt cggcctgtat aaagaaatca
     2461 ttccggaact ggcaagcaag aacctgaaaa atgattatcg tattctgagt cagaccctgt
     2521 gtgaactgtg tgataaaagc ccgaacctgt ttctgaagaa aaacgaacgt ctgcgcaaat
     2581 gcgttgaggt ggatattaac aatgcagata gcagcatgac ccgcaaatat cgtaattgta
     2641 ttgcacatct gacagttgtg cgcgaactga aagaatatat tggtgatatt cgtaccgtgg
     2701 acagctactt tagcatctat cattatgtta tgcagcggtg tattaccaaa cgcgaaaatg
     2761 ataccaaaca agaagagaaa atcaaatacg aggacgatct gcttaaaaac cacggctata
     2821 ccaaagattt tgtgaaagcc ctgaatagcc cgttcggtta taacattccg cgtttcaaaa
     2881 atctgagcat cgagcagctg tttgatcgta atgaatatct gacggaaaaa taataagaat
     2941 tcggatccct cgagttgaca gctagctcag tcctaggtat aatgctagca gagaccatgg
     3001 ccaccggtct ctgctaaaga tatgaggagc tcaaactcga gctgctaaca aagcccgaaa
     3061 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3121 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     3181 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3241 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3301 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3361 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3421 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3481 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3541 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3601 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     3661 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3721 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3781 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3841 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3901 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3961 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     4021 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4081 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     4141 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     4201 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4261 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4321 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4381 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4441 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4501 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4561 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     4621 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4681 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4741 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4801 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4861 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4921 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4981 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5041 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5101 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5161 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5221 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5281 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5341 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5401 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5461 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     5521 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     5581 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5641 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5701 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     5761 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5821 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5881 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5941 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     6001 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     6061 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     6121 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     6181 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     6241 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     6301 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     6361 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     6421 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     6481 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     6541 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     6601 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     6661 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6721 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     6781 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     6841 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     6901 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     6961 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     7021 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     7081 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     7141 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     7201 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     7261 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     7321 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     7381 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     7441 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     7501 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     7561 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     7621 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     7681 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     7741 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     7801 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     7861 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     7921 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     7981 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     8041 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     8101 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
