LOCUS Exported 5499 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of tagged protein in mammalian cells. ACCESSION . VERSION . KEYWORDS USH1CA1-TwinStrepII SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5499) TITLE Barr-Gillespie TwinStrep Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5499) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5499 /organism="synthetic DNA construct" /mol_type="other DNA" polyA_signal 1..49 /note="synthetic polyadenylation signal" misc_feature 63..154 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 103..122 /label=RVprimer3 /note="pGL3 vector, forward primer" protein_bind 190..210 /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" enhancer 271..574 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 575..778 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 729..749 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 799..823 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" regulatory 824..833 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2477..2566 /codon_start=1 /product="two Strep-Tag(R)II moieties connected by a linker for enhanced binding to Strep-Tactin(R), an engineered form of streptavidin (Schmidt et al., 2013)" /label=Twin-Strep-tag /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK" protein_bind complement(2570..2594) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" polyA_signal 2623..2744 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2634..2653) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2688..2707 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(2992..3009) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3163..3751) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3243..3262) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3922..4782) /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 4545..4564 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4783..4887) /gene="bla" /label=AmpR promoter rep_origin 4914..5369 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5001..5020) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5211..5232 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta 61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc 121 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcttacgcg 181 tgctagcatc aactttgtat agaaaagttg tagttattaa tagtaatcaa ttacggggtc 241 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 301 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 361 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 421 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 481 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 541 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 601 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 661 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 721 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg 781 tttagtgaac cgtcagatcc aagtttgtac aaaaaagcag ggcgccacca tggaccggaa 841 ggtggcccga gaattccgac acaaggtgga ttttctcatt gaaaatgacg cagagaagga 901 ctatctttat gatgtgctgc ggatgtatca ccagaccatg gatgtggctg tgctcgtggg 961 agacctgaag ctggtcatca atgaacccaa ccgcctgccg ctgtttgatg ccattcgacc 1021 cctgatccca ctgaagcacc aggtggagta tgaccagctg acaccccgac gctccaggaa 1081 actgaaggag gtacgcttgg accgtctgca cccagaaggt ctcggcctca gcgtgcgtgg 1141 aggcctggaa tttggctgtg gactctttat ctcccacctc atcaaaggtg gccaggcaga 1201 cagcgttggg cttcaggtag gggatgaaat tgtccggatc aacggctatt ccatctcttc 1261 ctgtacccat gaggaagtca tcaacctgat ccgcaccaag aagaccgtgt ccatcaaagt 1321 gagacacatc ggactgatcc ctgtgaagag ctctcctgag gagtccctca aatggcagta 1381 tgtggatcag ttcgtgtcgg aatctggggg tgtgcgaggt ggcttgggct cacctggcaa 1441 tcggacaacc aaggagaaga aggtgtttat cagtctagtg ggctctcggg gcctgggctg 1501 cagcatctcc agtggcccca tccagaagcc tggcatcttc gtcagccacg tgaagcctgg 1561 ctccctgtct gcagaggtgg ggttagagac aggagaccag attgtggaag tcaacggcat 1621 agacttcacc aacctggacc acaaggaggc tgtgaatgtc ctgaagagca gccgcagcct 1681 gaccatctcc atcgttgctg gagccggccg ggagctgttc atgacggacc gggaacggct 1741 ggaggaggca cggcagcgtg agctgcaacg gcaggaactc ctcatgcaga agcggctggc 1801 catggagtcc aacaagatcc tccaggagca gcaggagatg gagcgccaga ggagaaagga 1861 gatcgcccag aaggctgccg aggagaatga gagataccgg aaggagatgg aacagatctc 1921 ggaggaggaa gagaagttta agaagcagtg gaaagaagac tggggttcaa aggagcagct 1981 cattctgcct aagaccatca ccgcagaggt gcacccggtg ccccttcgca agcccaagta 2041 tgatcaggga gtggagccgg cagaccactt ggatggaagc acggaggagc agagacagca 2101 ggattttcga aaatacgaag aaggctttga tccctactcc atgttctcgc cagagcagat 2161 cgcagggaag gacgtccggc ttctgcgaat caagaaggag gggtctttag acttggccct 2221 ggagggaggc gtggactccc ccgtggggaa ggtggtcgtc tctgctgttt atgaaggggg 2281 agctgcggag aggcatggtg gcgttgtgaa aggggatgag atcatggcca tcaatggcaa 2341 gattgtgacg gactacaccc tggctgaggc cgaggctgcc ctacagaagg cctggaatca 2401 gggcggggac tggatagacc ttgtggttgc tgtctgtccc cctaaggagt atgatgacga 2461 gctaaccttc tttagatctg catggtccca tccgcaattt gagaaggggg gcggatctgg 2521 agggggcagt ggaggttccg cttggagcca cccccagttc gaaaagtgaa cccagctttc 2581 ttgtacaaag tggtgatggc cggccgcttc gagcagacat gataagatac attgatgagt 2641 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 2701 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 2761 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 2821 tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac 2881 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc 2941 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac 3001 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3061 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3121 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3181 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 3241 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 3301 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 3361 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 3421 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 3481 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 3541 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 3601 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 3661 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 3721 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 3781 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 3841 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 3901 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 3961 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 4021 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 4081 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 4141 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 4201 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 4261 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 4321 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 4381 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 4441 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 4501 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 4561 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 4621 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 4681 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 4741 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 4801 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 4861 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc 4921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 4981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 5041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 5101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 5161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 5221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 5281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 5341 ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg 5401 ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt 5461 aagtaatatt aaggtacggg aggtacttgg agcggccgc //