LOCUS       sequence-245680-        7025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Smad4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  AUTHORS   Moren A, Itoh S, Moustakas A, Dijke P, Heldin CH
  TITLE     Functional consequences of tumorigenic missense mutations in the 
            amino-terminal domain of Smad4.
  JOURNAL   Oncogene. 2000 Sep 7;19(38):4396-404.
  PUBMED    10980615
REFERENCE   2  (bases 1 to 7025)
  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: "Oncogene.";
            date: "2000-09-7"; volume: "19(38)"; pages: "4396-404"
COMMENT     Sequence Label: pcDNA3-Flag-Smad4
FEATURES             Location/Qualifiers
     source          1..7025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             688..711
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2416..2434)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2417..2434)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2454..2471)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2460..2684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2730..3158
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2817..2836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3027..3048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3167..3187)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3211..3421
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3233..3429
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3280..3415
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3342..3361
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3496..4290
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3550..3569)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4160..4179
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4464..4585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4501..4520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4555..4574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4634..4650)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4634..4650)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4647..4669)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4658..4674
                     /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(4682..4688,4689..4706,4707..4712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4727..4748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4865..4882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5036..5624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5116..5135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5795..6586,6587..6655))
                     /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     6418..6437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6656..6760)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6835..6854)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac cgccatggac tacaaggacg atgatgacaa gggcgaattc
      721 gagaatatgt ctattacgaa tacaccaaca agtaatgatg cctgtctgag cattgtgcat
      781 agtttgatgt gccatagaca aggtggagag agtgaaacat ttgcaaaaag agcaattgaa
      841 agtttggtaa agaagctgaa ggagaaaaaa gatgaattgg attctttaat aacagctata
      901 actacaaatg gagctcatcc tagtaaatgt gttaccatac agagaacatt ggatgggagg
      961 cttcaggtgg ctggtcggaa aggatttcct catgtgatct atgcccgtct ctggaggtgg
     1021 cctgatcttc acaaaaatga actaaaacat gttaaatatt gtcagtatgc gtttgactta
     1081 aaatgtgata gtgtctgtgt gaatccatat cactacgaac gagttgtatc acctggaatt
     1141 gatctctcag gattaacact gcagagtaat gctccatcaa gtatgatggt gaaggatgaa
     1201 tatgtgcatg actttgaggg acagccatcg ttgtccactg aaggacattc aattcaaacc
     1261 atccagcatc caccaagtaa tcgtgcatcg acagagacat acagcacccc agctctgtta
     1321 gccccatctg agtctaatgc taccagcact gccaactttc ccaacattcc tgtggcttcc
     1381 acaagtcagc ctgccagtat actggggggc agccatagtg aaggactgtt gcagatagca
     1441 tcagggcctc agccaggaca gcagcagaat ggatttactg gtcagccagc tacttaccat
     1501 cataacagca ctaccacctg gactggaagt aggactgcac catacacacc taatttgcct
     1561 caccaccaaa acggccatct tcagcaccac ccgcctatgc cgccccatcc cggacattac
     1621 tggcctgttc acaatgagct tgcattccag cctcccattt ccaatcatcc tgctcctgag
     1681 tattggtgtt ccattgctta ctttgaaatg gatgttcagg taggagagac atttaaggtt
     1741 ccttcaagct gccctattgt tactgttgat ggatacgtgg acccttctgg aggagatcgc
     1801 ttttgtttgg gtcaactctc caatgtccac aggacagaag ccattgagag agcaaggttg
     1861 cacataggca aaggtgtgca gttggaatgt aaaggtgaag gtgatgtttg ggtcaggtgc
     1921 cttagtgacc acgcggtctt tgtacagagt tactacttag acagagaagc tgggcgtgca
     1981 cctggagatg ctgttcataa gatctaccca agtgcatata taaaggtctt tgatttgcgt
     2041 cagtgtcatc gacagatgca gcagcaggcg gctactgcac aagctgcagc agctgcccag
     2101 gcagcagccg tggcaggaaa catccctggc ccaggatcag taggtggaat agctccagct
     2161 atcagtctgt cagctgctgc tggaattggt gttgatgacc ttcgtcgctt atgcatactc
     2221 aggatgagtt ttgtgaaagg ctggggaccg gattacccaa gacagagcat caaagaaaca
     2281 ccttgctgga ttgaaattca cttacaccgg gccctccagc tcctagacga agtacttcat
     2341 accatgccga ttgcagaccc acaaccttta gactgaggtc ttttaccgct cgagcatgca
     2401 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     2461 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2521 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2581 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2641 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2701 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2761 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2821 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2881 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2941 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3001 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3061 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3121 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3181 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3241 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3301 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3361 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3421 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3481 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3541 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3601 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3661 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3721 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3781 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3841 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3901 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3961 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4021 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4081 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4141 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4201 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4261 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4321 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4381 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4441 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4501 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4561 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4621 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4681 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4741 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4801 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4861 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4921 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4981 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5041 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5101 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5161 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5221 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5281 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5341 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5401 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5461 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5521 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5581 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5641 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5701 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5761 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6781 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     6841 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     6901 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     6961 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7021 gcgtt
//
