LOCUS       sequence-258299-        5356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5356)
  AUTHORS   Hata A, Lo RS, Wotton D, Lagna G, Massague J
  TITLE     Mutations increasing autoinhibition inactivate tumour suppressors 
            Smad2 and Smad4.
  JOURNAL   Nature. 1997 Jul 3. 388(6637):82-7.
  PUBMED    9214507
REFERENCE   2  (bases 1 to 5356)
  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"; journalName: "Nature. 
            1997 Jul 3. 388(6637):82-7."
COMMENT     Sequence Label: CS2 Flag-Smad2
FEATURES             Location/Qualifiers
     source          1..5356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        96..114
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     96..113
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             167..190
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1676..1810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1700..1719)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1754..1773
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1927..1947)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1927..1945)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1966..1982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1966..1982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1979..2001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1990..2006
                     /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(2014..2020,2021..2038,2039..2044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2059..2080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2197..2214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2368..2956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2448..2467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3127..3918,3919..3987))
                     /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     3750..3769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3988..4092)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4118..4573)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4255..4276)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4467..4486
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc gtttagggaa
       61 ccgccattct gcctggggac gtcggagcaa gcttgattta ggtgacacta tagaatacaa
      121 gctacttgtt ctttttgcag gatcccatcg attcgaattc accatggact acaaggacga
      181 cgatgacaag tcgtccatct tgccattcac gccgccagtt gtgaagagac tgctgggatg
      241 gaagaagtca gctggtgggt ctggaggagc aggcggagga gagcagaatg ggcaggaaga
      301 aaagtggtgt gagaaagcag tgaaaagtct ggtgaagaag ctaaagaaaa caggacgatt
      361 agatgagctt gagaaagcca tcaccactca aaactgtaat actaaatgtg ttaccatacc
      421 aagcacttgc tctgaaattt ggggactgag tacaccaaat acgatagatc agtgggatac
      481 aacaggcctt tacagcttct ctgaacaaac caggtctctt gatggtcgtc tccaggtatc
      541 ccatcgaaaa ggattgccac atgttatata ttgccgatta tggcgctggc ctgatcttca
      601 cagtcatcat gaactcaagg caattgaaaa ctgcgaatat gcttttaatc ttaaaaagga
      661 tgaagtatgt gtaaaccctt accactatca gagagttgag acaccagttt tgcctccagt
      721 attagtgccc cgacacaccg agatcctaac agaacttccg cctctggatg actatactca
      781 ctccattcca gaaaacacta acttcccagc aggaattgag ccacagagta attatattcc
      841 agaaacgcca cctcctggat atatcagtga agatggagaa acaagtgacc aacagttgaa
      901 tcaaagtatg gacacaggct ctccagcaga actatctcct actactcttt cccctgttaa
      961 tcatagcttg gatttacagc cagttactta ctcagaacct gcattttggt gttcaatagc
     1021 atattatgaa ttaaatcaga gggttggaga aaccttccat gcatcacagc cctcactcac
     1081 tgtagatggc tttacagacc catcaaattc agagaggttc tgcttaggtt tactctccaa
     1141 tgttaaccga aatgccacgg tagaaatgac aagaaggcat ataggaagag gagtgcgctt
     1201 atactacata ggtggggaag tttttgctga gtgcctaagt gatagtgcaa tctttgtgca
     1261 gagccccaat tgtaatcaga gatatggctg gcaccctgca acagtgtgta aaattccacc
     1321 aggctgtaat ctgaagatct tcaacaacca ggaatttgct gctcttctgg ctcagtctgt
     1381 taatcagggt tttgaagccg tctatcagct aactagaatg tgcaccataa gaatgagttt
     1441 tgtgaaaggg tggggagcag aataccgaag gcagacggta acaagtactc cttgctggat
     1501 tgaacttcat ctgaatggac ctctacagtg gttggacaaa gtattaactc agatgggatc
     1561 tccttcagtg cgttgctcaa gcatgtcata aagcttcacc aatcaagtcc catgaaaaga
     1621 cttaatgtaa caactcttct cgagcctcta gaactatagt gagtcgtatt acgtagatcc
     1681 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     1741 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     1801 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     1861 ggaggttttt taattcgcgg cgcgccgcgg cgccaatgca ttgggcccgg tacccagctt
     1921 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     1981 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     2041 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2101 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2161 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2221 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2281 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2341 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2401 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2461 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2521 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2581 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2641 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2701 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2761 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2821 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2881 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2941 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3001 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3061 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3121 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3181 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3241 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3301 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3361 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3421 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3481 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3541 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3601 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3661 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3721 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3781 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3841 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3901 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3961 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4021 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4081 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4141 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4201 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4261 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4321 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4381 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4441 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4501 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4561 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     4621 cgggcctctt cgctattacg ccagtcgatc gaccgccaat tcaatatggc gtatatggac
     4681 tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc gtatatggac
     4741 tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc tattggccag
     4801 gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt tttcctattg
     4861 acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct tcctaatacc
     4921 gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt cctattgacg
     4981 tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca attacggtaa
     5041 atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat tgacgtcaat
     5101 gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt caatgacggt
     5161 aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa gtacattact
     5221 attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg cggtaaatgg
     5281 cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca atgacgcaaa
     5341 tgggcgttcc attgac
//
