LOCUS       sequence-177474-        5989 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 5989)
  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 5989)
  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: pCMV5 Flag-Smad2 (1-456)
FEATURES             Location/Qualifiers
     source          1..5989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        504..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     658..678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             737..760
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    2136..2758
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2335..2354)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2782..2802)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2787..3116
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2967..3102
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3154..3173)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3155..3173)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3187..3203)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3187..3203)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3200..3222)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3211..3227
                     /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(3235..3241,3242..3259,3260..3265))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3280..3301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3418..3435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3589..4177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3669..3688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4348..5139,5140..5208))
                     /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     4971..4990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5209..5313)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5340..5795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5427..5446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5637..5658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5921..5943
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5935..5952
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5936..5952
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tagccatatt agtcattggt tatatagcat aaatcaatat tggctattgg ccattgcata
       61 cgttgtatct atatcataat atgtacattt atattggctc atgtccaata tgaccgccat
      121 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      181 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      241 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      301 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      361 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
      421 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
      481 tattagtcat cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat
      541 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      601 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc
      661 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc
      721 gtcagaattc accatggact acaaggacga cgatgacaag tcgtccatct tgccattcac
      781 gccgccagtt gtgaagagac tgctgggatg gaagaagtca gctggtgggt ctggaggagc
      841 aggcggagga gagcagaatg ggcaggaaga aaagtggtgt gagaaagcag tgaaaagtct
      901 ggtgaagaag ctaaagaaaa caggacgatt agatgagctt gagaaagcca tcaccactca
      961 aaactgtaat actaaatgtg ttaccatacc aagcacttgc tctgaaattt ggggactgag
     1021 tacaccaaat acgatagatc agtgggatac aacaggcctt tacagcttct ctgaacaaac
     1081 caggtctctt gatggtcgtc tccaggtatc ccatcgaaaa ggattgccac atgttatata
     1141 ttgccgatta tggcgctggc ctgatcttca cagtcatcat gaactcaagg caattgaaaa
     1201 ctgcgaatat gcttttaatc ttaaaaagga tgaagtatgt gtaaaccctt accactatca
     1261 gagagttgag acaccagttt tgcctccagt attagtgccc cgacacaccg agatcctaac
     1321 agaacttccg cctctggatg actatactca ctccattcca gaaaacacta acttcccagc
     1381 aggaattgag ccacagagta attatattcc agaaacgcca cctcctggat atatcagtga
     1441 agatggagaa acaagtgacc aacagttgaa tcaaagtatg gacacaggct ctccagcaga
     1501 actatctcct actactcttt cccctgttaa tcatagcttg gatttacagc cagttactta
     1561 ctcagaacct gcattttggt gttcaatagc atattatgaa ttaaatcaga gggttggaga
     1621 aaccttccat gcatcacagc cctcactcac tgtagatggc tttacagacc catcaaattc
     1681 agagaggttc tgcttaggtt tactctccaa tgttaaccga aatgccacgg tagaaatgac
     1741 aagaaggcat ataggaagag gagtgcgctt atactacata ggtggggaag tttttgctga
     1801 gtgcctaagt gatagtgcaa tctttgtgca gagccccaat tgtaatcaga gatatggctg
     1861 gcaccctgca acagtgtgta aaattccacc aggctgtaat ctgaagatct tcaacaacca
     1921 ggaatttgct gctcttctgg ctcagtctgt taatcagggt tttgaagccg tctatcagct
     1981 aactagaatg tgcaccataa gaatgagttt tgtgaaaggg tggggagcag aataccgaag
     2041 gcagacggta acaagtactc cttgctggat tgaacttcat ctgaatggac ctctacagtg
     2101 gttggacaaa gtattaactc agatgtaagg atcccgggtg gcatccctgt gacccctccc
     2161 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     2221 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     2281 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     2341 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     2401 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     2461 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     2521 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     2581 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt aaaataacta taccagcagg
     2641 aggacgtcca gacacagcat aggctacctg gccatgccca accggtggga catttgagtt
     2701 gcttgcttgg cactgtcctc tcatgcgttg ggtccactca gtagatgcct gttgaattgg
     2761 gtacgcggcc agcttggctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2821 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2881 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2941 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3001 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     3061 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3121 tcctcgagga actgaaaaac cagaaagtta attccctata gtgagtcgta ttaaattcgt
     3181 aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3241 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3301 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3361 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3421 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3481 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3541 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3601 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3661 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3721 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3781 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3841 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3901 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3961 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4021 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4081 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4141 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4201 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4261 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4321 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4381 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4441 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4501 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4561 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4621 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4681 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4741 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4801 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4861 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4921 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4981 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5041 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5101 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5161 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5221 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5281 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5401 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5521 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5581 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5641 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5701 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5761 cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac
     5821 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     5881 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     5941 acgacggcca gtgccaagct gatctataca ttgaatcaat attggcaat
//
