LOCUS       sequence-167595-        7098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Smad4 P130S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7098)
  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 7098)
  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 P130S
FEATURES             Location/Qualifiers
     source          1..7098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             922..945
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2650..2668)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2651..2668)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2688..2705)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2694..2918
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2964..3392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3051..3070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3261..3282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3401..3421)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3406..3736
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3587..3722
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3649..3668
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3803..4597
                     /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(3857..3876)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4467..4486
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4771..4892
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4808..4827)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4862..4881
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4941..4957)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4941..4957)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4954..4976)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4965..4981
                     /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(4989..4995,4996..5013,5014..5019))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5034..5055
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5172..5189)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5343..5931)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5423..5442)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6102..6893,6894..6962))
                     /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     6725..6744
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6963..7067)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccaccgccat ggactacaag gacgatgatg acaagggcga attcgagaat
      961 atgtctatta cgaatacacc aacaagtaat gatgcctgtc tgagcattgt gcatagtttg
     1021 atgtgccata gacaaggtgg agagagtgaa acatttgcaa aaagagcaat tgaaagtttg
     1081 gtaaagaagc tgaaggagaa aaaagatgaa ttggattctt taataacagc tataactaca
     1141 aatggagctc atcctagtaa atgtgttacc atacagagaa cattggatgg gaggcttcag
     1201 gtggctggtc ggaaaggatt tcctcatgtg atctatgccc gtctctggag gtggcctgat
     1261 cttcacaaaa atgaactaaa acatgttaaa tattgtcagt atgcgtttga cttaaaatgt
     1321 gatagtgtct gtgtgaattc atatcactac gaacgagttg tatcacctgg aattgatctc
     1381 tcaggattaa cactgcagag taatgctcca tcaagtatga tggtgaagga tgaatatgtg
     1441 catgactttg agggacagcc atcgttgtcc actgaaggac attcaattca aaccatccag
     1501 catccaccaa gtaatcgtgc atcgacagag acatacagca ccccagctct gttagcccca
     1561 tctgagtcta atgctaccag cactgccaac tttcccaaca ttcctgtggc ttccacaagt
     1621 cagcctgcca gtatactggg gggcagccat agtgaaggac tgttgcagat agcatcaggg
     1681 cctcagccag gacagcagca gaatggattt actggtcagc cagctactta ccatcataac
     1741 agcactacca cctggactgg aagtaggact gcaccataca cacctaattt gcctcaccac
     1801 caaaacggcc atcttcagca ccacccgcct atgccgcccc atcccggaca ttactggcct
     1861 gttcacaatg agcttgcatt ccagcctccc atttccaatc atcctgctcc tgagtattgg
     1921 tgttccattg cttactttga aatggatgtt caggtaggag agacatttaa ggttccttca
     1981 agctgcccta ttgttactgt tgatggatac gtggaccctt ctggaggaga tcgcttttgt
     2041 ttgggtcaac tctccaatgt ccacaggaca gaagccattg agagagcaag gttgcacata
     2101 ggcaaaggtg tgcagttgga atgtaaaggt gaaggtgatg tttgggtcag gtgccttagt
     2161 gaccacgcgg tctttgtaca gagttactac ttagacagag aagctgggcg tgcacctgga
     2221 gatgctgttc ataagatcta cccaagtgca tatataaagg tctttgattt gcgtcagtgt
     2281 catcgacaga tgcagcagca ggcggctact gcacaagctg cagcagctgc ccaggcagca
     2341 gccgtggcag gaaacatccc tggcccagga tcagtaggtg gaatagctcc agctatcagt
     2401 ctgtcagctg ctgctggaat tggtgttgat gaccttcgtc gcttatgcat actcaggatg
     2461 agttttgtga aaggctgggg accggattac ccaagacaga gcatcaaaga aacaccttgc
     2521 tggattgaaa ttcacttaca ccgggccctc cagctcctag acgaagtact tcataccatg
     2581 ccgattgcag acccacaacc tttagactga ggtcttttac cgctcgagca tgcatctaga
     2641 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     2701 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2761 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2821 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2881 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2941 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3001 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3061 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3121 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3181 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3241 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3301 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     3361 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3421 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     3481 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3541 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3601 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3661 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3721 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3781 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3841 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3901 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3961 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4021 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4081 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4141 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4201 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4261 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4321 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4381 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4441 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4501 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4561 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4621 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4681 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4741 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4801 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4861 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4921 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4981 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5041 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5101 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5161 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5221 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5281 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5341 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5401 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5461 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5521 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5581 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5641 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5701 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5761 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5821 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5881 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5941 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6001 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6061 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6121 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6181 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6241 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6301 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6361 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6421 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6481 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6541 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6601 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6661 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6721 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6781 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6841 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6901 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6961 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7021 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7081 aaagtgccac ctgacgtc
//
