LOCUS       Exported                8395 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  This lentiviral vector can be used to assay Cas9 activity..
ACCESSION   .
VERSION     .
KEYWORDS    pXPR_011
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8395)
  AUTHORS   Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, 
            Sullender M, Ebert BL, Xavier RJ, Root DE
  TITLE     Rational design of highly active sgRNAs for CRISPR-Cas9-mediated 
            gene inactivation.
  JOURNAL   Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026.
  PUBMED    25184501
REFERENCE   2  (bases 1 to 8395)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026."
FEATURES             Location/Qualifiers
     source          1..8395
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        7..507
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(519..538)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1015..1035
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1155..1220
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             1227..1949
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKS"
     primer_bind     complement(1272..1293)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1533..1552)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1880..1901
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1964..2552
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2017..2037)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2435..2446)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2624..2857
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2935..3056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2972..2991)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3026..3045
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3096..3231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3158..3177
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3251..3270)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3252..3270)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3280..3297)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3280..3296)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3289..3311)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3438..3893
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3525..3544)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3735..3756
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3919..4023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4024..4884
                     /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     complement(4242..4261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5055..5643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5544..5563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5797..5814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5931..5952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5967..5997
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6005..6021
                     /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)."
     primer_bind     6010..6032
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6029..6045
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6029..6045
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6064..6084
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6066..6084
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6112..6338
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6339..6519
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    6566..6691
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7184..7417
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7602..7646
                     /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"
     promoter        7824..8064
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     7824..7844
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7995..8014
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_feature    8226..8343
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"