LOCUS       Exported                8083 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd gen lentiviral vector for EGFP fusion; PGK driven puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    pLJM1-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8083)
  AUTHORS   Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled
            L, Sabatini DM
  TITLE     The Rag GTPases bind raptor and mediate amino acid signaling to 
            mTORC1.
  JOURNAL   Science. 2008 Jun 13. 320(5882):1496-501.
  PUBMED    18497260
REFERENCE   2  (bases 1 to 8083)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2008 Jun 13. 320(5882):1496-501."
FEATURES             Location/Qualifiers
     source          1..8083
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        290..493
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     444..464
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      532..541
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             538..1254
                     /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             538..1254
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(583..604)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(844..863)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1191..1212
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1325..1442
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1491..2001
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1920..1938
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2023..2622
                     /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(2023..2042)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2519..2539
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2750..2983
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3055..3176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3092..3111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3146..3165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3216..3351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3278..3297
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3371..3390)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3372..3390)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3400..3417)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3400..3416)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3409..3431)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3558..4013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3645..3664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3855..3876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4039..4143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4144..5004
                     /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(4362..4381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5175..5763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5664..5683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5917..5934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6051..6072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6087..6117
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6125..6141
                     /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     6130..6152
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6149..6165
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6149..6165
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6184..6204
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6186..6204
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6232..6458
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6459..6639
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    6686..6811
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7304..7537
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7722..7766
                     /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"
     primer_bind     join(8064..8083,1)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"