LOCUS       Exported               12607 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses PspCas13b in mammalian cells for knockdown of target RNAs 
            in combination with compatible guide RNAs. Localized to the 
            cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    pC0046-EF1a-PspCas13b-NES-HIV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12607)
  AUTHORS   Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung 
            J, Zhang F
  TITLE     RNA editing with CRISPR-Cas13.
  JOURNAL   Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
  PUBMED    29070703
REFERENCE   2  (bases 1 to 12607)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180."
FEATURES             Location/Qualifiers
     source          1..12607
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2445..3623
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2676..3614
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3571..3591
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6918..6944
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             6954..7034
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     misc_feature    7062..7650
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7115..7135)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7533..7544)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7653..7669)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7654..7670)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8175..8355
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(8381..8398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8387..8611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8657..9085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8744..8763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8954..8975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9094..9114)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9099..9428
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9279..9414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9341..9360
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9476..9523
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9542..9916
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10046..10167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10083..10102)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10137..10156
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10216..10232)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10216..10232)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10229..10251)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10240..10256
                     /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        complement(10264..10294)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10309..10330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10447..10464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10618..11206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10698..10717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11377..12237)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     12000..12019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12238..12342)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12417..12436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"