LOCUS       Exported                8771 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd gen lentiviral eGFP expression vector, CMV/TO promoter, Puro .
ACCESSION   .
VERSION     .
KEYWORDS    pLenti CMV/TO eGFP Puro (w159-1)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8771)
  AUTHORS   Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, 
            Campisi J, Yaswen P, Cooper PK, Kaufman PD.
  TITLE     A versatile viral system for expression and depletion of proteins in
            mammalian cells.
  JOURNAL   PLoS One. 2009 Aug 6;4(8):e6529.
  PUBMED    19657394
REFERENCE   2  (bases 1 to 8771)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2009-08-6"; volume: "4(8)"; pages: "e6529"
FEATURES             Location/Qualifiers
     source          1..8771
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             132..992
                     /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(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1905..1922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2039..2060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2075..2105
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2113..2129
                     /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     2118..2140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2137..2153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2137..2153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2172..2192
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2174..2192
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     LTR             2448..2628
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    2672..2797
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3290..3523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3707..3751
                     /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    4012..4129
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        4152..4455
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4456..4659
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4610..4630
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4656..4680
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4764..4788
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      4848..4857
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4854..5573
                     /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             4854..5570
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4899..4920)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5160..5179)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5507..5528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5598..5622)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    5653..6241
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5706..5726)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6124..6135)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        6255..6754
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6290..6313)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6687..6706
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6775..7374
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6775..6794)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7271..7291
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             7512..7745
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7817..7938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7854..7873)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7908..7927
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7957..8092
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8019..8038
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(8124..8143)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8125..8143)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8153..8170)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8153..8169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8162..8184)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8314..8769
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8401..8420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8611..8632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"