LOCUS       Exported                8467 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Conditional overexpression vector. Deletion of dsRed by Cre 
            recombinase results in the rapid loss of dsRed and the activation of
            your gene fused to eGFP expression..
ACCESSION   .
VERSION     .
KEYWORDS    pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8467)
  AUTHORS   Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, 
            Clevers H
  TITLE     Controlled gene expression in primary Lgr5 organoid cultures.
  JOURNAL   Nat Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802.
  PUBMED    22138822
REFERENCE   2  (bases 1 to 8467)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802."
FEATURES             Location/Qualifiers
     source          1..8467
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..517
                     /label=5 LTR
                     /note="5 long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1439..1472
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      1501..1510
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1507..2184
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(1687..1707)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1905..1924
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2099..2122
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    2209..2242
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      2293..2302
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2299..3018
                     /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             2299..3015
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2344..2365)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2605..2624)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2952..2973
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        3028..3527
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3063..3086)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3460..3479
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3548..4147
                     /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(3548..3567)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4044..4064
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4217..4805
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4270..4290)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4688..4699)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4862..5376
                     /label=3 LTR
                     /note="3 long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5538..5554
                     /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(5562..5592)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5607..5628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5745..5762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5916..6504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5996..6015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6675..7535)
                     /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     7298..7317
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7536..7640)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7708..7726
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7764..7786)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     7886..7905
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8099..8121
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8249..8268
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"