LOCUS       40924_32325        8376 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8376)
  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 8376)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8376)
  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..8376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             341..516
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell
                     virus"
     CDS             988..1404
                     /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    complement(1417..1450)
                     /label=lox2272
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             1467..2063
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MVEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             2094..2159
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             2175..2849
                     /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(2863..2896)
                     /label=lox5171
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     ncRNA           complement(2961..3002)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse
                     miR-155 microRNA (Uva et al., 2013)"
     ncRNA           complement(3066..3092)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
     CDS             complement(3155..3871)
                     /codon_start=1
                     /label=EmGFP
                     /note="Emerald GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    3885..3918
                     /label=lox2272
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3965..3998
                     /label=lox5171
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    4044..4632
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             4689..5204
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem
                     cell virus"
     primer_bind     complement(5373..5389)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5397..5413)
                     /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(5421..5451)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5466..5487)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(5604..5621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5775..6363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6537..7394)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7395..7499)
                     /label=AmpR promoter
     primer_bind     7567..7585
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(7623..7645)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7745..7764
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     7958..7980
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8156..8175
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt agatccataa cttcgtatag gataccttat
     1441 acgaagttat ctcaggtacc gccaccatgg tggagtacaa gcccacggtg cgcctcgcca
     1501 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     1561 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     1621 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     1681 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     1741 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     1801 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     1861 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     1921 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     1981 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     2041 gcatgacccg caagcccggt gccctgtaca agaaacagaa aattgtggca ccagtgaaac
     2101 agactttgaa ttttgacctt ctcaagttgg cgggagacgt cgagtccaac cctgggcccg
     2161 gcccggtcgc caccatggat agcactgaga acgtcatcaa gcccttcatg cgcttcaagg
     2221 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggca
     2281 agccctacga gggcacccag accgccaagc tgcaggtgac caagggcggc cccctgccct
     2341 tcgcctggga catcctgtcc ccccagttcc agtacggctc caaggtgtac gtgaagcacc
     2401 ccgccgacat ccccgactac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
     2461 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     2521 gcaccttcat ctaccacgtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa
     2581 tgcagaagaa gactctgggc tgggagccct ccaccgagcg cctgtacccc cgcgacggcg
     2641 tgctgaaggg cgagatccac aaggcgctga agctgaaggg cggcggccac tacctggtgg
     2701 agttcaagtc aatctacatg gccaagaagc ccgtgaagct gcccggctac tactacgtgg
     2761 actccaagct ggacatcacc tcccacaacg aggactacac cgtggtggag cagtacgagc
     2821 gcgccgaggc ccgccaccac ctgttccagt agtctagaag ccataacttc gtatagtaca
     2881 cattatacga agttatgcat acctggagga aaaaaaaaag gggagagtca ggggtttaaa
     2941 ctcgagtgcg gccagatctg ggccatttgt tccatgtgag tgctagtaac aggccttgtg
     3001 tcctgacaac aggcagcata atgagtcagt cagtggccaa aactcattat ggactgcctg
     3061 ttgtcagcat acagccttca gcaagcctcc aggatccact ggtcgactca ctacctccct
     3121 ttaaacgatc gacggccacg aagtgcttag cttacttgta cagctcgtcc atgccgagag
     3181 tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
     3241 ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt
     3301 cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
     3361 tgtggcgggt cttgaagttc accttgatgc cgttcttctg cttgtcggcg gtgatataga
     3421 ccttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga
     3481 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     3541 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt
     3601 cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cgggcgaagc
     3661 actgcacgcc gtaggtgaag gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     3721 tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg
     3781 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     3841 cggtgaacag ctcctcgccc ttgctcacca tggtggcgac cggtataact tcgtataagg
     3901 tatcctatac gaagttatcc attcaggctg tgctagcatc aatggcatgg cacaaagctt
     3961 agccataact tcgtataatg tgtactatac gaagttatcc cgggttaaac gacctgcagc
     4021 caagcttatc gataattccc gataatcaac ctctggatta caaaatttgt gaaagattga
     4081 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     4141 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     4201 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     4261 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     4321 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     4381 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc
     4441 tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     4501 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4561 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4621 ccgcctcccc gcatcgggaa ttatcgataa aataaaagat tttatttagt ctccagaaaa
     4681 aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt
     4741 gcaaggcatg gaaaatacat aactgagaat agagaagttc agatcaaggt taggaacaga
     4801 gagacagcag aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     4861 gggccaagaa cagatggtcc ccagatgcgg tcccgccctc agcagtttct agagaaccat
     4921 cagatgtttc cagggtgccc caaggacctg aaaatgaccc tgtgccttat ttgaactaac
     4981 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
     5041 cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt
     5101 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg
     5161 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa cagtttcttg
     5221 aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct gactcaatta
     5281 gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat ggacagcgca
     5341 gaagagctgg ggagaattaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5401 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5461 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5521 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5581 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5641 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5701 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5761 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5821 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5881 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5941 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6001 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6061 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6121 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6181 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6241 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6301 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6361 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6421 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6481 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6541 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6601 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6661 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6721 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6781 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6841 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6901 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6961 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7021 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7081 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7141 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7201 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7261 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7321 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7381 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7441 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7501 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7561 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     7621 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     7681 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     7741 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     7801 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     7861 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     7921 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     7981 acggcgcaag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     8041 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     8101 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     8161 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     8221 gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc
     8281 tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
     8341 aaaagatttt atttagtctc cagaaaaagg ggggaa
//
