LOCUS       Exported                8543 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  measures Cas9 activity.
ACCESSION   .
VERSION     .
KEYWORDS    pXPR_047
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8543)
  TITLE     David Root CRISPR plasmids
REFERENCE   2  (bases 1 to 8543)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8543
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    69..90
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        105..135
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    143..159
                     /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     148..170
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     167..183
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     167..183
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     202..222
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        204..222
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        250..476
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             477..657
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    704..829
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1322..1555
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1740..1784
                     /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        1962..2202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1962..1982
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2133..2152
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_feature    2364..2481
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2540..3040
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2959..2977
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(3052..3071)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3548..3568
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3688..3753
                     /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             3760..4476
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3760..4476
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3805..3826)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4066..4085)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4413..4434
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4645..5233
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4698..4718)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5116..5127)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5305..5538
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5616..5737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5653..5672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5707..5726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5777..5912
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5839..5858
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5932..5951)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5933..5951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5961..5978)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5961..5977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5970..5992)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6119..6574
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6206..6225)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6416..6437
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6600..6704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6705..7565
                     /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(6923..6942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7736..8324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8225..8244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8478..8495
                     /label=L4440
                     /note="L4440 vector, forward primer"