LOCUS Exported 7118 bp ds-DNA circular SYN 31-AUG-2021 DEFINITION Expresses fluorescently tagged human Smad3 in mammalian cells.. ACCESSION . VERSION . KEYWORDS mNeonGreen-Smad3 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 Aug 31, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7118 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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" 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 3352..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(3397..3418) /label=EGFP-N /note="EGFP, reverse primer" 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(6000..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 //