LOCUS       Exported                7556 bp ds-DNA     circular SYN 20-DEC-2021
DEFINITION  Expresses MBP fused to Cas14a1.
ACCESSION   .
VERSION     .
KEYWORDS    pLBH531_MBP-Cas14a1 expression
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7556)
  AUTHORS   Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, 
            Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA
  TITLE     Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.
  JOURNAL   Science. 2018 Oct 18. pii: science.aav4294. doi: 
            10.1126/science.aav4294.
  PUBMED    30337455
REFERENCE   2  (bases 1 to 7556)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2018 Oct 18. pii: science.aav4294. doi: 10.1126/science.aav4294."
FEATURES             Location/Qualifiers
     source          1..7556
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    93..232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     148..167
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(247..264)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(418..1006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(498..517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1177..2037)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1800..1819
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2038..2142)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2210..2228
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2255..2283
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      2649..2696
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2692..2710
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4432..4452)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(4507..5607)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(4510..4533)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(5617..5646)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             5666..5688
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(5776..5795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5777..5795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5879..5898)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             7356..7547
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(join(7541..7556,1..7))
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"