LOCUS       Exported                8475 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  An empty sgRNA expression vector for expression of sgRNA for Sp Cas9
            (3rd generation lentiviral vector).
ACCESSION   .
VERSION     .
KEYWORDS    pLenti SpBsmBI sgRNA Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8475)
  AUTHORS   Pham H, Kearns NA, Maehr R
  TITLE     Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian 
            Cells.
  JOURNAL   Methods Mol Biol. 2016;1358:43-57. doi: 10.1007/978-1-4939-3067-8_3.
  PUBMED    26463376
REFERENCE   2  (bases 1 to 8475)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2016"; volume: "1358:43-57. doi"; pages: " 
            10.1007/978-1-4939-3067-8_3"
FEATURES             Location/Qualifiers
     source          1..8475
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(20..40)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(20..38)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(59..75)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(59..75)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(72..94)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    83..99
                     /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(107..137)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    152..173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(290..307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(461..1049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(541..560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1220..2080)
                     /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     1843..1862
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2081..2185)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2211..2666)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2348..2369)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2560..2579
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2793..2815
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2807..2824
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2808..2824
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2834..2853
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2834..2852
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2873..3008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2927..2946)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3048..3169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3059..3078)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3113..3132
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             3241..3474
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3561..4149
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3667..3678
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4076..4096
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        complement(4778..5959)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4787..5729
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4810..4830)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    6164..6280
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6311..6551
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6311..6331
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6482..6501
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6579..6654
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(6933..6977)
                     /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    7162..7395
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    7888..8013
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             8060..8240
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     promoter        complement(8241..8467)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"