LOCUS       sequence-272945-        4237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORF insert for replicon MoClo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4237)
  AUTHORS   Wagner TE, Becraft JR, Bodner K, Teague B, Zhang X, Woo A, Porter E,
            Alburquerque B, Dobosh B, Andries O, Sanders NN, Beal J, Densmore D,
            Kitada T, Weiss R
  TITLE     Small-molecule-based regulation of RNA-delivered circuits in 
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2018 Nov;14(11):1043-1050. doi: 
            10.1038/s41589-018-0146-9. Epub 2018 Oct 16.
  PUBMED    30327560
REFERENCE   2  (bases 1 to 4237)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41589-018-0146-9"; journalName: "Nat Chem Biol"; date: 
            "2018-11"; volume: "14"; issue: "11"; pages: "1043-1050"
COMMENT     Sequence Label: pTW927
FEATURES             Location/Qualifiers
     source          1..4237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(146..469)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    396..452
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(465..481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(465..481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(478..500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    489..505
                     /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(join(513..519,520..537,538..543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    558..579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1335..1391
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1392..2102
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(2247..2264)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2418..3006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2498..2517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3177..3968,3969..4037))
                     /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     3800..3819
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4038..4142)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4210..4228
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      421 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
      481 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
      541 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
      601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      661 agaggcggtt tgcgtattgg gcgctcttcc gctggtctca caccatgtca agactcgaca
      721 agagcaaggt gattaacagt gcactggaac ttctcaatga agttgggatc gaggggctga
      781 ctactagaaa actcgcacag aaactggggg ttgagcagcc caccttgtac tggcacgtta
      841 aaaacaaaag ggccctgctg gatgctctgg ccatcgagat gctggatagg catcataccc
      901 acttctgccc tctggaagga gaatcctggc aggatttcct tagaaacaac gccaagtcct
      961 ttcgctgtgc ccttcttagc caccgggatg gtgctaaagt ccatctcggc acacgaccaa
     1021 ctgagaagca gtacgaaact ctcgagaacc agctggcctt tctctgtcaa cagggctttt
     1081 ctcttgaaaa cgccctgtac gcactgagtg cagttgggca ctttacactc ggatgtgttc
     1141 tggaggacca agaacatcag gtggcaaagg aagagaggga gacacctacg actgactcca
     1201 tgccccctct cttgaggcag gcaatagaat tgttcgacca tcagggcgca gaacccgcct
     1261 ttctgtttgg gctggaactg attatctgcg gtcttgagaa acagctgaag tgcgagtccg
     1321 ggagcggaag cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga
     1381 accctggacc tatggtgtct aagggcgagg agctgattaa ggagaacatg cacatgaagc
     1441 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     1501 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     1561 tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca
     1621 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
     1681 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     1741 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     1801 tgcagaagaa aacactcggc tgggaggcct ccaccgagat gctgtacccc gctgacggcg
     1861 gcctggaagg cagaagcgac atggccctga agctcgtggg cgggggccac ctgatctgca
     1921 acttgaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     1981 actatgtgga cagaagactg gaaagaatca aggaggccga caaagagaca tacgtcgagc
     2041 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
     2101 ataagcttag ccatggcttc ccgccggagg tggaggagca ggatgatggc acgctgccca
     2161 tgtcttgtgc ccaggagagc gggatggacc gtcaccctgc agcctgtgct tctgctagga
     2221 tcaatgtgta ataatgagac cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2281 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2341 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2401 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2461 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2521 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2581 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2641 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2701 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2761 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2821 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2881 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2941 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3001 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3061 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3121 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3181 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3241 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3301 gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag
     3361 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3421 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3481 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3541 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3601 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3661 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3721 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3781 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3841 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3901 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3961 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4021 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4081 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4141 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4201 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
