LOCUS       Exported                8248 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses E. coli codon optimized EsCas13d in the pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    pET28a-MH6-EsCas13d
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)
  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."
FEATURES             Location/Qualifiers
     source          1..8248
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     promoter        53..82
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             102..124
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             156..173
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3054..3088
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(3183..3201)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3197..3244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3281..3736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3368..3387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3578..3599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3828..4643)
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4551..4570
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4635..4654
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4765..5353
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5254..5273
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5507..5524
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5539..5678
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5604..5623)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5764..5786
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5780..5971)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6780..7862)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7824..7843
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7863..7940)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8146..8165
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"