LOCUS       Exported                7477 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Lentiviral expression of AAVS1-targeting sgRNA; insert can be 
            replaced with custom sgRNA (see protocol).
ACCESSION   .
VERSION     .
KEYWORDS    pLX-sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  AUTHORS   Wang T, Wei JJ, Sabatini DM, Lander ES
  TITLE     Genetic screens in human cells using the CRISPR-Cas9 system.
  JOURNAL   Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981. 
            Epub 2013 Dec 12.
  PUBMED    24336569
REFERENCE   2  (bases 1 to 7477)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.1246981"; journalName: "Science"; date: "2014-01-3-
            3"; volume: "343"; issue: "6166"; pages: "80-4"
FEATURES             Location/Qualifiers
     source          1..7477
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        12..238
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             239..419
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    466..591
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1084..1317
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1502..1546
                     /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        1721..2221
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2140..2158
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2233..2628
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    2688..2805
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2923..3163
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2923..2943
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3094..3113
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3192..3267
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3279..3303)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3341..3929
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3394..3414)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3812..3823)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4001..4234
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4312..4433
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4349..4368)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4403..4422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4473..4608
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4535..4554
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4628..4647)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4629..4647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4657..4674)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4657..4673)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4666..4688)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4815..5270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4902..4921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5112..5133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5296..5400
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5401..6261
                     /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(5619..5638)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6432..7020
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6921..6940
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7174..7191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7308..7329
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7344..7374
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7382..7398
                     /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     7387..7409
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7406..7422
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7406..7422
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7441..7461
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7443..7461
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"