LOCUS       Exported               10457 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral transfer plasmid. Expresses EGFP and 
            puromycin resistance in mammalian cells under the control of a 
            doxycycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    FUW-tetO-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10457)
  AUTHORS   Panciera T, Azzolin L, Fujimura A, Di Biagio D, Frasson C, Bresolin 
            S, Soligo S, Basso G, Bicciato S, Rosato A, Cordenonsi M, Piccolo S
  TITLE     Induction of Expandable Tissue-Specific Stem/Progenitor Cells 
            through Transient Expression of YAP/TAZ.
  JOURNAL   Cell Stem Cell. 2016 Sep 9. pii: S1934-5909(16)30256-9. doi: 
            10.1016/j.stem.2016.08.009.
  PUBMED    27641305
REFERENCE   2  (bases 1 to 10457)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem 
            Cell. 2016 Sep 9. pii: S1934-5909(16)30256-9. doi: 
            10.1016/j.stem.2016.08.009."
FEATURES             Location/Qualifiers
     source          1..10457
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        408..606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     561..581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             624..804
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    851..976
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1469..1702
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1887..1931
                     /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    2229..2346
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2399..2669
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        2702..2740
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2737..2761
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2895..2904
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2901..3620
                     /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             2901..3617
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2946..2967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3207..3226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3554..3575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3660..4246
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3827..3844)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4054..4073
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4247..4846
                     /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(4247..4266)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4743..4763
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4938..5526
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4991..5011)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5409..5420)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5529..5545)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5530..5546)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6051..6231
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6257..6274)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6263..6487
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6533..6961
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6620..6639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6830..6851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6970..6990)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6975..7304
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7155..7290
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7217..7236
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7352..7399
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7418..7792
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7922..8043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7959..7978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8013..8032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8092..8108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8092..8108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8105..8127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8116..8132
                     /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)."
     promoter        complement(8140..8170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8185..8206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8323..8340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8494..9082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8574..8593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9253..10113)
                     /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     9876..9895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10114..10218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10293..10312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"