LOCUS       Exported                9542 bp ds-DNA     circular SYN 06-APR-2021
DEFINITION  Twinstrep-SUMO-huLwCas13a for recombinant protein bacterial 
            expression. Insert is human codon optimized but expresses well in 
            bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    Twinstrep-SUMO-huLwCas13a
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9542)
  AUTHORS   Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung 
            J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, 
            Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, 
            Collins JJ, Zhang F
  TITLE     Nucleic acid detection with CRISPR-Cas13a/C2c2.
  JOURNAL   Science. 2017 Apr 13. pii: eaam9321. doi: 10.1126/science.aam9321.
  PUBMED    28408723
REFERENCE   2  (bases 1 to 9542)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9542
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(170..192)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     misc_feature    278..417
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     333..352
                     /label=pRS-marker
                     /note="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(432..449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(603..1191)
                     /direction=LEFT
                     /label=ori
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(683..702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1362..2222)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1985..2004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2223..2327)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="bla promoter"
     primer_bind     2395..2413
                     /label=pBRforEco
                     /note="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2440..2468
                     /label=tet promoter
                     /note="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      2643..2690
                     /label=T7 terminator
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2686..2704
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2754..6209)
                     /codon_start=1
                     /product="Leptotrichia wadei CRISPR-Cas effector that acts 
                     as an RNA-guided RNAse (Abudayyeh et al., 2017)"
                     /label=LwCas13a
                     /note="previously called C2c2"
                     /translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
                     IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
                     DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
                     NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
                     YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
                     KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
                     NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
                     NENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE
                     IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
                     IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
                     SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
                     KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
                     SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
                     TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
                     ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
                     IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
                     GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
                     IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
                     IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
                     IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
     CDS             complement(6216..6506)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="S. cerevisiae SMT3 (truncated)"
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     CDS             complement(6507..6530)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /note="Strep-Tag II"
                     /translation="WSHPQFEK"
     CDS             complement(6567..6590)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /note="Strep-Tag II"
                     /translation="WSHPQFEK"
     CDS             complement(6603..6620)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(6630..6647)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="6xHis"
                     /translation="HHHHHH"
     RBS             6666..6688
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6703..6727
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac repressor encoded by lacI 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)."
     primer_bind     complement(6727..6746)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6728..6746)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6830..6849)
                     /label=pBRrevBam
                     /note="pBRrevBam"
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7055..7132
                     /gene="lacI"
                     /label=lacI promoter
                     /note="lacI promoter"
     CDS             7133..8215
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="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     complement(7152..7171)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             9527..176
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"