LOCUS       sequence-244251-        4810 bp    DNA     circular SYN 14-APR-2025
DEFINITION  ORF insert for replicon MoClo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4810)
  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 4810)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pJB332
FEATURES             Location/Qualifiers
     source          1..4810
                     /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."
     primer_bind     complement(2820..2837)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2991..3579)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3071..3090)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3750..4541,4542..4610))
                     /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     4373..4392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4611..4715)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4783..4801
                     /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 ggagcggcgg tggcggtggc ggtggcggtg gtatgagcac agctcgcacc gagaacccgg
     1381 tgattatggg cctgtccagc cagaacggac agctcagagg gcctgtaaag gcttcagcag
     1441 gccccggcgg aggcggcaca caaccacaac cacagcttaa tcagcttaag aatactagca
     1501 ctattaataa cggaacaccg cagcaggccc aaagcatggc tgccacaatt aaacccggag
     1561 atgactggaa gaagaccctg aagctccctc caaaagatct caggattaaa actagcgatg
     1621 ttacttcaac aaagggaaat gagttcgaag actactgtct gaagcgagag ttgctgatgg
     1681 ggattttcga aatgggctgg gagaagccct ctcctattca agaagagagc atccccatcg
     1741 ctctgtccgg gagggacatc cttgccaggg ctaaaaatgg gaccggaaaa tcaggagctt
     1801 acttgatccc actccttgaa aggcttgatc tcaagaagga caacatccaa gctatggtta
     1861 tcgtgccaac tagagaactc gccctccagg tcagccagat ttgcatccag gtgagtaagc
     1921 acatgggcgg agctaaggtg atggctacaa ctggagggac taacctgcga gacgacataa
     1981 tgagacttga tgacacagtc catgtggtca tcgctacacc tgggaggatt ctggatctga
     2041 tcaaaaaagg agtggcaaag gtggatcatg tgcagatgat agtcttggac gaggccgaca
     2101 aactgctgag ccaagacttt gtgcagatca tggaggatat catcttgaca ctccccaaga
     2161 accgacagat tctgctgtac tccgcaacat ttcctctttc cgttcagaaa ttcatgaact
     2221 cacatctcca gaaaccttat gagatcaatt tgatggaaga actgacactg aagggcgtga
     2281 cccagtatta tgcctacgtt actgagaggc aaaaggtcca ctgcctgaat actctcttct
     2341 ccaggctcca gatcaaccag tctatcatct tttgcaatag ctcccagcga gtcgagctgc
     2401 tggctaagaa gatctcacag cttggatatt cctgtttcta catccatgct aagatgagac
     2461 aagagcacag aaaccgcgtc tttcatgatt tccggaacgg actctgtcgc aacctggttt
     2521 gcacagatct ttttactaga ggcatcgata tccaagcagt gaacgtggtt atcaacttcg
     2581 actttcccaa actcgccgag acttatcttc atagaattgg ccgatccggt aggtttgggc
     2641 acctggggct cgccatcaat ctcattacgt atgatgatag gttcaacctc aagtcaatag
     2701 aagagcagtt ggggaccgag atcaaaccaa tcccgagcaa tattgacaaa tcactctatg
     2761 tggccgaata ccattcagag cctgccgagg atgagaagcc ttaataatga gaccgcttcc
     2821 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2881 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2941 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3001 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3061 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3121 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3181 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3241 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3301 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3361 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3421 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3481 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3541 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3601 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3661 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3721 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3781 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3841 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3901 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3961 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4021 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4081 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4141 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4201 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4261 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4321 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4381 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4441 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4501 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4561 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4621 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4681 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4741 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4801 ccctttcgtc
//
