LOCUS       40924_32345        8467 bp 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    .
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)
  TITLE     Direct Submission
REFERENCE   3  (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."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /codon_start=1
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
                     /translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
                     NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
                     PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
     protein_bind    1439..1472
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             1507..2181
                     /codon_start=1
                     /label=DsRed-Express2
                     /note="noncytotoxic tetrameric variant of DsRed fluorescent
                     protein (Strack et al., 2008)"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     protein_bind    complement(2209..2242)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2299..3015
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        3028..3527
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             3548..4144
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    4217..4805
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             4862..5376
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem
                     cell virus"
     protein_bind    complement(5538..5554)
                     /label=lac operator
                     /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    complement(5607..5628)
                     /label=CAP binding site
                     /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"
     CDS             complement(6678..7535)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7536..7640)
                     /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"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatcacta gtccagtgtg gtggaattat
     1441 aacttcgtat agcatacatt atacgaagtt ataattctgc agatatccag cacagtggcg
     1501 gccaccatgg atagcactga gaacgtcatc aagcccttca tgcgcttcaa ggtgcacatg
     1561 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg caagccctac
     1621 gagggcaccc agaccgccaa gctgcaggtg accaagggcg gccccctgcc cttcgcctgg
     1681 gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac
     1741 atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     1801 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcaccttc
     1861 atctaccacg tgaagttcat cggcgtgaac ttcccctccg acggccccgt aatgcagaag
     1921 aagactctgg gctgggagcc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag
     1981 ggcgagatcc acaaggcgct gaagctgaag ggcggcggcc actacctggt ggagttcaag
     2041 tcaatctaca tggccaagaa gcccgtgaag ctgcccggct actactacgt ggactccaag
     2101 ctggacatca cctcccacaa cgaggactac accgtggtgg agcagtacga gcgcgccgag
     2161 gcccgccacc acctgttcca gtagagctag ctgaataagg ccgctcgaat aacttcgtat
     2221 agcatacatt atacgaagtt attcgagtct agagggcccg ttagatctct cgaggttaac
     2281 gaattcgact acgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2341 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2401 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2461 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2521 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2581 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2641 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2701 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2761 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2821 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2881 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2941 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3001 gacgagctgt acaagtaaaa ttctaccggg taggggaggc gcttttccca aggcagtctg
     3061 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
     3121 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
     3181 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
     3241 gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg
     3301 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
     3361 ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca
     3421 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     3481 gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcagcccaa
     3541 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3601 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3661 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3721 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3781 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3841 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3901 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     3961 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     4021 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     4081 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     4141 tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     4201 atcgataatt cccgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     4261 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4321 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4381 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4441 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4501 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4561 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     4621 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     4681 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4741 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4801 cccgcatcgg gaattatcga taaaataaaa gattttattt agtctccaga aaaagggggg
     4861 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     4921 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
     4981 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     5041 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
     5101 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
     5161 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
     5221 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa
     5281 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     5341 tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct tgaagttgga
     5401 gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat tagccactgt
     5461 tttgaatcca catactccaa tactcctgaa atagttcatt atggacagcg cagaaagagc
     5521 tggggagaat tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5581 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5641 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5701 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5761 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5821 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5881 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5941 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6001 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6061 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6121 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6181 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6241 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6301 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6361 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6421 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6481 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6541 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6601 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6661 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6721 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6781 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6841 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6901 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6961 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7021 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7081 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7141 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7201 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7261 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7321 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7381 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7441 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7501 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7561 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7621 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7681 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     7741 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     7801 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     7861 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     7921 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg
     7981 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     8041 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     8101 cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc atgcaaggag atggcgccca
     8161 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     8221 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     8281 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggcga ttagtccaat
     8341 ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa tatcaccagc
     8401 tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt ctccagaaaa
     8461 agggggg
//
