LOCUS       Exported                9846 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Lentiviral vector pDG2i-Flag-Puro, complete sequence.
ACCESSION   LT009444
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9846)
  AUTHORS   De Groote P, Grootjans S, Lippens S, Eichperger C, Leurs K, Kahr I, 
            Tanghe G, Bruggeman I, De Schamphelaire W, Urwyler C, Vandenabeele 
            P, Haustraete J, Declercq W.
  TITLE     Generation of a new Gateway-compatible inducible lentiviral vector 
            platform allowing easy derivation of co-transduced cells
  JOURNAL   BioTechniques 60 (5), 252-259 (2016)
  PUBMED    27177818
REFERENCE   2  (bases 1 to 9846)
  AUTHORS   De Schamphelaire W.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-DEC-2015) BCCM/LMBP, Universiteit Gent, 
            Technologiepark 927, 9052, BELGIUM
REFERENCE   3  (bases 1 to 9846)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9846
                     /organism="Lentiviral vector pDG2i-Flag-Puro"
                     /mol_type="other DNA"
                     /note="BCCM/LMBP Plasmid collection (Ghent University, 
                     Belgium) accession number LMBP 7944."
                     /db_xref="taxon:1783451"
     misc_feature    75..90
                     /label=HIV-1 cPPT
                     /note="HIV-1 cPPT"
     regulatory      91..325
                     /regulatory_class="terminator"
                     /note="RSV/HIV-1 dU3/3 LTR"
     LTR             92..325
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    397..518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      complement(397..471)
                     /regulatory_class="terminator"
                     /note="SV40 polyA late"
     regulatory      397..471
                     /regulatory_class="terminator"
                     /note="SV40 polyA early"
     rep_origin      558..693
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      607..682
                     /label=SV40 ori
                     /note="SV40 ori"
     promoter        complement(714..732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      complement(716..732)
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     primer_bind     complement(742..758)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      900..1355
                     /label=f1 origin
                     /note="f1 origin"
     promoter        1381..1485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1486..2346
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="ampicillin resistance"
                     /protein_id="CVH74069.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            1486..2346
                     /gene="bla"
                     /label=bla
     rep_origin      2517..3105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      2887..3289
                     /label=pMB1 ori
                     /note="pMB1 ori"
     regulatory      3381..3464
                     /regulatory_class="promoter"
                     /note="lac promoter"
     protein_bind    3393..3414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3429..3459
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3467..3483
                     /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     3491..3507
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3528..3546
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      3528..3544
                     /regulatory_class="promoter"
                     /note="T3 promoter"
     promoter        3574..3800
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     regulatory      3653..3981
                     /regulatory_class="promoter"
                     /note="RSV/HIV-1 5 LTR"
     LTR             3801..3981
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    4028..4153
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4092..4136
                     /label=HIV-1 psi packaging signal
                     /note="HIV-1 psi packaging signal"
     misc_feature    4646..4879
                     /label=HIV-1 Rev response element
                     /note="HIV-1 Rev response element"
     protein_bind    5396..5414
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      5397..5664
                     /regulatory_class="promoter"
                     /note="hCMVTRE promoter"
     protein_bind    5431..5449
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5467..5485
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5503..5521
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5538..5556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5574..5592
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        5604..5642
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      complement(5707..5831)
                     /regulatory_class="attenuator"
                     /note="gateway recombination site attR1"
     protein_bind    5707..5831
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        5856..5886
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5940..6599
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyl transferase"
                     /label=cat
                     /note="chloramphenicol resistance"
                     /protein_id="CVH74070.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            5940..6599
                     /gene="cat"
                     /label=cat
     CDS             6941..7246
                     /codon_start=1
                     /gene="ccdB"
                     /product="ccdB"
                     /label=ccdB
                     /note="B gene of the control of cell death locus of the 
                     Escherichia coli F plasmid"
                     /protein_id="CVH74071.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            6941..7246
                     /gene="ccdB"
                     /label=ccdB
     regulatory      7287..7411
                     /regulatory_class="attenuator"
                     /note="gateway recombination site attR2"
     protein_bind    complement(7287..7411)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    7455..7478
                     /label=FLAG epitope
                     /note="FLAG epitope"
     CDS             7455..7478
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7514..7796
                     /regulatory_class="promoter"
                     /note="SV40 early promoter"
     promoter        7523..7853
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7704..7839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      7753..7828
                     /label=SV40 ori
                     /note="SV40 ori"
     CDS             7905..8612
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     CDS             7905..8603
                     /codon_start=1
                     /gene="rtTA3"
                     /product="Reverse tetracycline-responsive transcriptional 
                     activator"
                     /label=rtTA3
                     /protein_id="CVH74073.1"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDML"
     gene            7905..8603
                     /gene="rtTA3"
                     /label=rtTA3
     misc_feature    8613..8646
                     /label=loxP
                     /note="loxP"
     protein_bind    8613..8646
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACAT). loxP511 sites are compatible with each
                     other, but incompatible with wild-type loxP sites."
     regulatory      8669..9241
                     /regulatory_class="ribosome_binding_site"
                     /note="5 UTR of the EMCV polyprotein gene (IRES)"
     misc_feature    8669..9241
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             9244..9846
                     /codon_start=1
                     /gene="pac"
                     /product="puromycin N-acetyl-transferase"
                     /label=pac
                     /note="puromycin resistance gene"
                     /protein_id="CVH74074.1"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGSRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     gene            9244..9846
                     /gene="pac"
                     /label=pac