LOCUS       sequence-341509-        7118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fluorescently tagged human Smad3 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7118)
  AUTHORS   Frick CL, Yarka C, Nunns H, Goentoro L
  TITLE     Sensing relative signal in the Tgf-beta/Smad pathway.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Apr 4;114(14):E2975-E2982. doi: 
            10.1073/pnas.1611428114. Epub 2017 Mar 20.
  PUBMED    28320972
REFERENCE   2  (bases 1 to 7118)
  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"; doi: 
            "10.1073/pnas.1611428114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-04-4- 4"; volume: "114"; issue: "14"; pages: 
            "E2975-E2982"
COMMENT     Sequence Label: mNeonGreen-Smad3
FEATURES             Location/Qualifiers
     source          1..7118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             615..1322
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      870..879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2643..2764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2654..2673)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2708..2727
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2958..3315
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2958..3149
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2981..3001)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3166..3301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3228..3247
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(3352..3354,3355..3357,3358..4068)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3658..3677)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4005..4026
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4072..4092
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4096..4116
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4120..4140
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4156..4212
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4213..4812
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    4837..4885
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5041..5058)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5212..5800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5292..5311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6000..6791,6792..6860))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    6965..7013
                     /note="synthetic polyadenylation signal"
     misc_feature    7027..7118
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7067..7086
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gccggtacct agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      601 gtcagatcag atctatggtg agcaagggcg aggaggataa catggcctct ctcccagcga
      661 cacatgagtt acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg
      721 gcaccggcaa tccaaatgat ggttatgagg agttaaacct gaagtccacc aagggtgacc
      781 tccagttctc cccctggatt ctggtccctc atatcgggta tggcttccat cagtacctgc
      841 cctaccctga cgggatgtcg cctttccagg ccgccatggt agatggctcc ggataccaag
      901 tccatcgcac aatgcagttt gaagatggtg cctcccttac tgttaactac cgctacacct
      961 acgagggaag ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg
     1021 gtcctgtgat gaccaactcg ctgaccgctg cggactggtg caggtcgaag aagacttacc
     1081 ccaacgacaa aaccatcatc agtaccttta agtggagtta caccactgga aatggcaagc
     1141 gctaccggag cactgcgcgg accacctaca cctttgccaa gccaatggcg gctaactatc
     1201 tgaagaacca gccgatgtac gtgttccgta agacggagct caagcactcc aagaccgagc
     1261 tcaacttcaa ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca
     1321 agaagctttc gtccatcctg cctttcactc ccccgatcgt gaagcgcctg ctgggctgga
     1381 agaagggcga gcagaacggg caggaggaga aatggtgcga gaaggcggtc aagagcctgg
     1441 tcaagaaact caagaagacg gggcagctgg acgagctgga gaaggccatc accacgcaga
     1501 acgtcaacac caagtgcatc accatcccca ggtccctgga tggccggttg caggtgtccc
     1561 atcggaaggg gctccctcat gtcatctact gccgcctgtg gcgatggcca gacctgcaca
     1621 gccaccacga gctgcgggcc atggagctgt gtgagttcgc cttcaatatg aagaaggacg
     1681 aggtctgcgt gaatccctac cactaccaga gagtagagac accagttcta cctcctgtgt
     1741 tggtgccacg ccacacagag atcccggccg agttcccccc actggacgac tacagccatt
     1801 ccatccccga aaacactaac ttccccgcag gcatcgagcc ccagagcaat attccagaga
     1861 ccccaccccc tggctacctg agtgaagatg gagaaaccag tgaccaccag atgaaccaca
     1921 gcatggacgc aggttctcca aacctatccc cgaatccgat gtccccagca cataataact
     1981 tggacctgca gccagttacc tactgcgagc cggccttctg gtgctccatc tcctactacg
     2041 agctgaacca gcgcgtcggg gagacattcc acgcctcgca gccatccatg actgtggatg
     2101 gcttcaccga cccctccaat tcggagcgct tctgcctagg gctgctctcc aatgtcaaca
     2161 ggaatgcagc agtggagctg acacggagac acatcggaag aggcgtgcgg ctctactaca
     2221 tcggagggga ggtcttcgca gagtgcctca gtgacagcgc tatttttgtc cagtctccca
     2281 actgtaacca gcgctatggc tggcacccgg ccaccgtctg caagatccca ccaggatgca
     2341 acctgaagat cttcaacaac caggagttcg ctgccctcct ggcccagtcg gtcaaccagg
     2401 gctttgaggc tgtctaccag ttgacccgaa tgtgcaccat ccgcatgagc ttcgtcaaag
     2461 gctggggagc ggagtacagg agacagactg tgaccagtac cccctgctgg attgagctgc
     2521 acctgaatgg gcctttgcag tggcttgaca aggtcctcac ccagatgggc tccccaagca
     2581 tccgctgttc cagtgtgtct tagtctagag tcggggcggc cggccgcttc gagcagacat
     2641 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     2701 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     2761 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     2821 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtttgcgta
     2881 ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg aaataacctc tgaaagagga
     2941 acttggttag ctaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3001 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3061 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3121 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3181 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3241 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3301 ctaggctttt gcaaaaagct cgattcttct gacactagcg ccaccgctag catggtgagc
     3361 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3421 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3481 accctgaagt tcatcagcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3541 accctgacct ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     3601 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3661 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3721 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3781 tacaacgcca tcagcgacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag
     3841 gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3901 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3961 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     4021 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga tccaaaaaag
     4081 aagagaaagg tagatccaaa aaagaagaga aaggtagatc caaaaaagaa gagaaaggta
     4141 ctcgagggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag
     4201 aaccctggac ctatgaccga gtacaagcct accgtgcgcc tggccactcg cgatgatgtg
     4261 ccccgcgccg tccgcactct ggccgccgct ttcgccgact accccgctac ccggcacacc
     4321 gtggaccccg accggcacat cgagcgtgtg acagagttgc aggagctgtt cctgacccgc
     4381 gtcgggctgg acatcggcaa ggtgtgggta gccgacgacg gcgcggccgt ggccgtgtgg
     4441 actacccccg agagcgttga ggccggcgcc gtgttcgccg agatcggccc ccgaatggcc
     4501 gagctgagcg gcagccgcct ggccgcccag cagcaaatgg agggcctgct tgccccccat
     4561 cgtcccaagg agcctgcctg gtttctggcc actgtaggag tgagccccga ccaccagggc
     4621 aagggcttgg gcagcgccgt cgtgttgccc ggcgtagagg ccgccgaacg cgccggtgtg
     4681 cccgcctttc tcgaaacaag cgcaccaaga aaccttccat tctacgagcg cctgggcttc
     4741 accgtgaccg ccgatgtcga ggtgcccgag ggacctagga cctggtgtat gacacgaaaa
     4801 cctggcgcct aatgatctag aaccggtcat ggccgcaata aaatatcttt attttcatta
     4861 catctgtgtg ttggtttttt gtgtgttcga actagatgct gtcgaccgat gcccttgaga
     4921 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     4981 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc
     5041 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5101 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5161 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5221 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5281 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5341 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5401 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5461 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5521 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5581 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5641 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5701 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5761 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5821 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5881 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5941 aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca ctgcagtggt
     6001 taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg ttcgtccata
     6061 gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc atcaggcccc
     6121 agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc agcaatgaac
     6181 cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc ctccatccag
     6241 tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag tttccgaaga
     6301 gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat ggcttcgttc
     6361 aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg aagaaatgca
     6421 gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt gttgtcgctc
     6481 atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag atgcttttcc
     6541 gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg accaagctgc
     6601 tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt gaaagtgctc
     6661 atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct attgagatcc
     6721 agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac tttcaccagc
     6781 gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat gagtgcgaca
     6841 cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt attgaagcat ttatcagggt
     6901 tactagtacg tctctcaagg ataagtaagt aatattaagg tacgggaggt attggacagg
     6961 ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat
     7021 agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc
     7081 tgtccccagt gcaagtgcag gtgccagaac atttctct
//
