LOCUS       Exported                8798 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd gen lentiviral eGFP expression vector, CMV promoter, Neo.
ACCESSION   .
VERSION     .
KEYWORDS    pLenti CMV GFP Neo (657-2)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8798)
  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 8798)
  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..8798
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        58..162
                     /gene="bla"
                     /label=AmpR promoter
     CDS             163..1023
                     /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(381..400)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1194..1782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1683..1702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1936..1953
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2070..2091
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2106..2136
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2144..2160
                     /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     2149..2171
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2168..2184
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2168..2184
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2203..2223
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2205..2223
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2251..2477
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2478..2658
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    2705..2830
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3323..3556
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3741..3785
                     /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    4052..4168
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        4191..4494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4495..4698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4649..4669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4695..4719
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4788..4797
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4794..5513
                     /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             4794..5510
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4839..4860)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5100..5119)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5447..5468
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5529..6117
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5582..5602)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6000..6011)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        6160..6659
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6195..6218)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6592..6611
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6673..7476
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6736..6755)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7346..7365
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             7567..7800
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7872..7993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7909..7928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7963..7982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8033..8168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8095..8114
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(8188..8207)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8189..8207)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8217..8234)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8217..8233)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8226..8248)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(8375..8798,1..32)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8462..8481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8672..8693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"