LOCUS Exported 8003 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses pentamerised full-length protein ectodomain with no N-linked glycans in mammalian cells. Contains a C-terminal rat Cd4 d3+4 tag, COMP pentamerisation domain, flag- and 6xHis tags. . ACCESSION . VERSION . KEYWORDS TRSP-COMP-blac-flag-his SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8003) TITLE Pentamerised full-length protein ectodomains with no N-linked glycans REFERENCE 2 (bases 1 to 8003) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8003 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 310..329 /label=pMT2-F /note="Synthetic intron, forward primer" regulatory 352..361 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1537..2322 /codon_start=1 /gene="bla(M)" /product="beta-lactamase lacking the signal sequence" /label=bla(M) /note="allows cytosolic expression of beta-lactamase" /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATT LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW" CDS 2329..2394 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2401..2418 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 2454..2509 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(2508..2527) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(4745..4763) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4831..4935 /gene="bla" /label=AmpR promoter CDS 4936..5796 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 5967..6555 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6456..6475 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6709..6726 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6843..6864 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6879..6909 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6917..6933 /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)." primer_bind 6922..6944 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6941..6957 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6941..6957 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 7228..7607 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7608..7811 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7762..7782 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 7808..7832 /label=LNCX /note="Human CMV promoter, forward primer" ORIGIN 1 ctcgagaaag gcgtctaacc agtcacagtc gcaaggtagg ctgagcaccg tggcgggcgg 61 cagcgggtgg cggtcggggt tgtttctggc ggaggtgctg ctgatgatgt aattaaagta 121 ggcggtcttg agacggcgga tggtcgaggt gaggtgtggc aggcttgaga tccagctgtt 181 ggggtgagta ctccctctca aaagcgggca ttacttctgc gctaagattg tcagtttcca 241 aaaacgagga ggatttgata ttcacctggc ccgatctggc catacacttg agtgacaatg 301 acatccactt tgcctttctc tccacaggtg tccactccca ggtccaagtt tgccaccatg 361 gagtttcaga cccaggtact catgtccctg ctgctctgca tgtctggtgc ggccgccgac 421 ttcttcctgc ggtatcggac cggcttcatc agaagccggc tggaaaccta catcggcaac 481 agcgacgtgc ggtacaacaa ggccttcatc aacaaccggc tgctgaacga gcacgcccac 541 tgcgacgctt ggagcgagtg gtccgcctgc agcaagacct gcgactacgg catcaagatc 601 agagtgaaga tcagcaccga ccagaccaag agcaaggcct gctccaatat cgccgagagc 661 accatctgcc acgagcacat ctgtccccgg accttcgagg aagccgagga aacatacctg 721 cacaacaaag agaaagagaa gaagaaaaag ttccggacca ccggcgcgcc gtcgacctcc 781 atcacggcct ataagagtga gggggagtca gcggagttct ccttcccact caaccttgga 841 gaggaaagcc tgcagggaga gttgagatgg aaggcagaga aggctccttc ttcccagtcc 901 tggatcacct tctccctaaa gaaccaaaag gtgtctgtgc agaagtctac tagcaacccc 961 aagttccagc tgtccgaaac gctcccactc acccttcaga taccccaggt ctcccttcag 1021 tttgctggtt ctggcaacct gaccctgact ctggacagag ggatactgta tcaggaagtg 1081 aacctggtgg tgatgaaagt gactcagccc gacagcaaca ctttgacctg tgaggtgatg 1141 ggacccacct cacccaagat gagactgatc ttgaagcagg agaatcagga ggccagggtc 1201 tccaggcagg agaaagtgat tcaagtgcag gcccctgaag caggggtgtg gcaatgtcta 1261 ctgagtgaag gtgaagaggt caagatggac tccaagatcc aggttttatc caaagggttg 1321 aattccggcg gcggctccgg aggcggtacc ggtggcggcg acctagcccc acagatgctt 1381 cgagaactcc aggagactaa tgcggcgctg caagacgtga gagagctctt gcgacagcag 1441 gtcaaggaga tcaccttcct gaagaatacg gtgatggaat gtgacgcctg cggtctagac 1501 agaaacctgc ctccactagc acctctgggc ccacacccag aaacgctggt gaaagtaaaa 1561 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 1621 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 1681 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 1741 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 1801 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 1861 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 1921 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 1981 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 2041 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 2101 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 2161 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 2221 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 2281 agacagatcg ctgagatagg tgcctcactg attaagcatt ggggccctga ctacaaagac 2341 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caagggccct 2401 caccaccacc atcaccacta ataataagga tcccccgacc tcgacctctg gctaataaag 2461 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac 2521 atatgggagg gcaaatcatt tggtcgagat ccctcggaga tctctagcta gaggatcgat 2581 ccccgccccg gacgaactaa acctgactac gacatctctg ccccttcttc gcggggcagt 2641 gcatgtaatc ccttcagttg gttggtacaa cttgccaact gggccctgtt ccacatgtga 2701 cacggggggg gaccaaacac aaaggggttc tctgactgta gttgacatcc ttataaatgg 2761 atgtgcacat ttgccaacac tgagtggctt tcatcctgga gcagactttg cagtctgtgg 2821 actgcaacac aacattgcct ttatgtgtaa ctcttggctg aagctcttac accaatgctg 2881 ggggacatgt acctcccagg ggcccaggaa gactacggga ggctacacca acgtcaatca 2941 gaggggcctg tgtagctacc gataagcgga ccctcaagag ggcattagca atagtgttta 3001 taaggccccc ttgttaaccc taaacgggta gcatatgctt cccgggtagt agtatatact 3061 atccagacta accctaattc aatagcatat gttacccaac gggaagcata tgctatcgaa 3121 ttagggttag taaaagggtc ctaaggaaca gcgatatctc ccaccccatg agctgtcacg 3181 gttttattta catggggtca ggattccacg agggtagtga accattttag tcacaagggc 3241 agtggctgaa gatcaaggag cgggcagtga actctcctga atcttcgcct gcttcttcat 3301 tctccttcgt ttagctaata gaataactgc tgagttgtga acagtaaggt gtatgtgagg 3361 tgctcgaaaa caaggtttca ggtgacgccc ccagaataaa atttggacgg ggggttcagt 3421 ggtggcattg tgctatgaca ccaatataac cctcacaaac cccttgggca ataaatacta 3481 gtgtaggaat gaaacattct gaatatcttt aacaatagaa atccatgggg tggggacaag 3541 ccgtaaagac tggatgtcca tctcacacga atttatggct atgggcaaca cataatccta 3601 gtgcaatatg atactggggt tattaagatg tgtcccaggc agggaccaag acaggtgaac 3661 catgttgtta cactctattt gtaacaaggg gaaagagagt ggacgccgac agcagcggac 3721 tccactggtt gtctctaaca cccccgaaaa ttaaacgggg ctccacgcca atggggccca 3781 taaacaaaga caagtggcca ctcttttttt tgaaattgtg gagtgggggc acgcgtcagc 3841 ccccacacgc cgccctgcgg ttttggactg taaaataagg gtgtaataac ttggctgatt 3901 gtaaccccgc taaccactgc ggtcaaacca cttgcccaca aaaccactaa tggcaccccg 3961 gggaatacct gcataagtag gtgggcgggc caagataggg gcgcgattgc tgcgatctgg 4021 aggacaaatt acacacactt gcgcctgagc gccaagcaca gggttgttgg tcctcatatt 4081 cacgaggtcg ctgagagcac ggtgggctaa tgttgccatg ggtagcatat actacccaaa 4141 tatctggata gcatatgcta tcctaatcta tatctgggta gcataggcta tcctaatcta 4201 tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaattta 4261 tatctgggta gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta 4321 tatctgggta gtatatgcta tcctaatctg tatccgggta gcatatgcta tcctaataga 4381 gattagggta gtatatgcta tcctaattta tatctgggta gcatatacta cccaaatatc 4441 tggatagcat atgctatcct aatctatatc tgggtagcat atgctatcct aatctatatc 4501 tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc 4561 tgggtagtat atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc 4621 tgggtagcat atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc 4681 cgggtagcat atgctatcct catgataagc tgtcaaacat gagaattttc ttgaagacga 4741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 4801 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 4861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 4921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 4981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 5041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 5101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 5161 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat 5221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 5281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 5341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 5401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 5461 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa 5521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 5581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 5641 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 5701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 5761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 5821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 5881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 5941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 6061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 6121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 6181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 6241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 6301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 6361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 6421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 6481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 6541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 6601 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 6661 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 6721 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 6781 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 6841 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 6901 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat 6961 gattacgcca agctctagct agaggtcgac caattctcat gtttgacagc ttatcatcgc 7021 agatccgggc aacgttgttg ccattgctgc aggcgcagaa ctggtaggta tggaagatct 7081 atacattgaa tcaatattgg caattagcca tattagtcat tggttatata gcataaatca 7141 atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg 7201 gctcatgtcc aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat 7261 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 7321 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 7381 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 7441 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg 7501 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact 7561 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 7621 ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 7681 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 7741 gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 7801 taagcagagc tcgtttagtg aaccgtcaga tcctcactct cttccgcatc gctgtctgcg 7861 agggccagct gttgggctcg cggttgagga caaactcttc gcggtctttc cagtactctt 7921 ggatcggaaa cccgtcggcc tccgaacggt actccgccac cgagggacct gagcgagtcc 7981 gcatcgaccg gatcggaaaa cct //