LOCUS Exported 5955 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION For mammalian expression of the human NODAL open reading frame (with a mutated Cysteine residue) with an internal MYC tag. ACCESSION . VERSION . KEYWORDS NODAL-matMYC-C312S SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5955) AUTHORS Findlay SD, Bilyk O, Lypka K, Waskiewicz AJ, Postovit L TITLE Genetically regulated human NODAL splice variants are differentially post-transcriptionally processed and functionally distinct JOURNAL bioRxiv 276170 REFERENCE 2 (bases 1 to 5955) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5955 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 141..520 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 521..724 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 675..695 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 750..769 /label=T7 /note="T7 promoter, forward primer" promoter 750..768 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1541..1570 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 1919..1948 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 1967..1990 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" primer_bind complement(2010..2026) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2010..2026) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" polyA_signal 2037..2659 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2236..2255) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" rep_origin complement(2808..3396) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2888..2907) /label=pBR322ori-F /note="pBR322 origin, forward primer" polyA_signal 3725..3772 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind 3797..3816 /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" CDS complement(4004..4798) /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4115..4134) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 4725..4744 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(4833..5190) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4847..4982 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(4901..4920) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 5147..5167 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter complement(5192..5296) /gene="bla" /label=AmpR promoter rep_origin 5323..5778 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5410..5429) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5620..5641 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5904..5926 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5918..5935 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5919..5935 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gaatcaatat tggcaattag ccatattagt cattggttat atagcataaa tcaatattgg 61 ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg 121 tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 181 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa 421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac 481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 541 caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 601 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa 661 ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 721 agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc ggccgggaat 781 tcatcgactg gatccggtac cgaggagatc tgccgccgcg atcgccatgc acgcccactg 841 cctgcccttc cttctgcacg cctggtgggc cctactccag gcgggtgctg cgacggtggc 901 cactgcgctc ctgcgtacgc gggggcagcc ctcgtcgcca tcccctctgg cgtacatgct 961 gagcctctac cgcgacccgc tgccgagggc agacatcatc cgcagcctac aggcagaaga 1021 tgtggcagtg gatgggcaga actggacgtt tgcttttgac ttctccttcc tgagccaaca 1081 agaggatctg gcatgggctg agctccggct gcagctgtcc agccctgtgg acctccccac 1141 tgagggctca cttgccattg agattttcca ccagccaaag cccgacacag agcaggcttc 1201 agacagctgc ttagagcggt ttcagatgga cctattcact gtcactttgt cccaggtcac 1261 cttttccttg ggcagcatgg ttttggaggt gaccaggcct ctctccaagt ggctgaagca 1321 ccctggggcc ctggagaagc agatgtccag ggtagctgga gagtgctggc cgcggccccc 1381 cacaccgcct gccaccaatg tgctccttat gctctactcc aacctctcgc aggagcagag 1441 gcagctgggt gggtccacct tgctgtggga agccgagagc tcctggcggg cccaggaggg 1501 acagctgtcc tgggagtggg gcaagaggca ccgtcgacat gagcagaaac tcatctcaga 1561 agaggatctg catcacttgc cagacagaag tcaactgtgt cggaaggtca agttccaggt 1621 ggacttcaac ctgatcggat ggggctcctg gatcatctac cccaagcagt acaacgccta 1681 tcgctgtgag ggcgagtgtc ctaatcctgt tggggaggag tttcatccga ccaaccatgc 1741 atacatccag agtctgctga aacgttacca gccccaccga gtcccttcca cttcttgtgc 1801 cccagtgaag accaagccgc tgagcatgct gtatgtggat aatggcagag tgctcctaga 1861 tcaccataaa gacatgatcg tggaagaatg tgggtgcctc tgacgtacgc ggccgctcga 1921 gcagaaactc atctcagaag aggatctggc agcaaatgat atcctggatt acaaggatga 1981 cgacgataag gtttaaacgg ccggccgcgg tcatagctgt ttcctgaaca gatcccgggt 2041 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 2101 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 2161 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct 2221 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca 2281 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg 2341 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt 2401 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc 2461 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt 2521 taaaataact ataccagcag gaggacgtcc agacacagca taggctacct ggccatgccc 2581 aaccggtggg acatttgagt tgcttgcttg gcactgtcct ctcatgcgtt gggtccactc 2641 agtagatgcc tgttgaattg ggtacgcggc cagcggcgag cggtatcagc tcactcaaag 2701 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2761 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2821 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2881 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2941 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3001 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3061 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3121 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3181 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3241 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3301 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3361 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3421 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3481 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3541 atatatgagt aacctgaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct 3601 gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg 3661 ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt 3721 ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg 3781 cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccct agggtgggcg 3841 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg 3901 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg 3961 ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa 4021 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga 4081 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt 4141 cctgatagcg atccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat 4201 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt 4261 cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt 4321 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc 4381 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca 4441 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct 4501 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca 4561 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca 4621 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg 4681 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga 4741 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc 4801 gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc tccaaaaaag 4861 cctcctcact acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata 4921 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg 4981 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata 5041 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc 5101 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac 5161 acattccaca gctggttctt tccgcctcag gactcttcct ttttcaatat tattgaagca 5221 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 5281 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg 5341 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 5401 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 5461 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 5521 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 5581 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 5641 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5701 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5761 tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc 5821 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 5881 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa 5941 gctgatctat acatt //