LOCUS       Exported               11269 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Lentiviral vector for constitutive expression of the LaG17 GFP 
            nanobody synNotch TetRVP64 Receptor (SFFV promoter).
ACCESSION   .
VERSION     .
KEYWORDS    pHR_SFFV_LaG17_synNotch_TetRVP64
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11269)
  AUTHORS   Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim 
            WA
  TITLE     Engineering Customized Cell Sensing and Response Behaviors Using 
            Synthetic Notch Receptors.
  JOURNAL   Cell. 2016 Feb 11;164(4):780-91. doi: 10.1016/j.cell.2016.01.012. 
            Epub 2016 Jan 28.
  PUBMED    26830878
REFERENCE   2  (bases 1 to 11269)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2016.01.012"; journalName: "Cell"; date: 
            "2016-02-11- 11"; volume: "164"; issue: "4"; pages: "780-91"
FEATURES             Location/Qualifiers
     source          1..11269
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             85..318
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(343..361)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(344..361)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(651..670)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(884..903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1003..1025
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1063..1081)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1149..1253
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1254..2114
                     /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(1472..1491)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2285..2873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2774..2793
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3027..3044
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3114..3134)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3119..3448
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3299..3434
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3361..3380
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     intron          4582..4647
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4777..4797
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5222..5356
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5259..5278)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5313..5332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5524..6157
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    6204..6329
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6826..7059
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7244..7288
                     /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    7573..7690
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7843..8250
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      8318..8327
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             8387..8416
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    9779..9799
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             9800..10546
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    10656..11244
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10709..10729)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11127..11138)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11247..11263)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11248..11264)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"