LOCUS Exported 10549 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 SIAP-1-COMP-blac-flag-his SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10549) TITLE Pentamerised full-length protein ectodomains with no N-linked glycans REFERENCE 2 (bases 1 to 10549) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10549 /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 4081..4866 /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 4873..4938 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4945..4962 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 5000..5055 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(5054..5073) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(7291..7309) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7377..7481 /gene="bla" /label=AmpR promoter CDS 7482..8342 /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 8513..9101 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9002..9021 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9255..9272 /label=L4440 /note="L4440 vector, forward primer" protein_bind 9389..9410 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9425..9455 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9463..9479 /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 9468..9490 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9487..9503 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9487..9503 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 9774..10153 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 10154..10357 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 10308..10328 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 10354..10378 /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 ggccgcctac 421 aacatcaaga gcggcaacac cgccaacaat atcaaatacg tgaacgtgct ggacaacgac 481 cgggacatca acacccacag cgtgctgccc gaggtggaaa acgtgatcga gcggaaggac 541 atctaccggc agatcaactt catggaaacc ttcgtgtcca gcaacaacat gatgcacgac 601 agagagaagc acaccagcaa cgacgccggc agctacgaga tcaccggcat cgtggacggc 661 atgaagatcg gccaccccat cagcgtggcc ctgggcagcc agtacagcaa ctacttcgac 721 tacctgcaga tcgtgcacct ggactacacc aacagccggt tcagcttcac cgtgggcgag 781 ggcaagtact acctgcggac ctacggcagc acctacatga cccctagcgc catcaagatc 841 aaggtgccct gcgagaagtg caagttcatc aacagcgagt acagcggcat catcaagatt 901 atcccttacg agacaaacaa caacctgttc atctacaact gggtgctgca gaccagcagc 961 cctctggccc tggaaaacat caataccgtg ttcagcgacg aggccgacct gatccacggc 1021 aacagcctga gcgaggaatt caagatcgac agcagcgccg ctgccaccag cctgaacacc 1081 ttctatggca tcgtgctgca cggcatctgg tcctccgagt acgccgagcg gctgctgacc 1141 gtgatctccg agttccccga ctgcgtgaag atgagcgccc acgacaagaa cgccagaagc 1201 aagcagcgga agaaccagaa gtggatcctg gtcaacgagg acctgggctc cttcgacatg 1261 aagatggaag tgtgcgagga agtgaactgc gactactccg ccatcatcca cgtgtccaag 1321 cacgccttcg agtactccaa gaaactggtg cacaaccggg gcagaaacgg ccggtactac 1381 agcagacggg tggaaaagat cctgatccgg gccctgctga gcctggactt cagcctgttt 1441 atcacctact tccagcagaa acacggcgtg accctgctgg accctcagta cgactacgag 1501 ctgatcacca acatggccgg ctacagctcc aacaactacc agagctggaa ccacaacctg 1561 gaagaactgg tggaactggc caccagctgg gacgagtacc ccaagggcct gcagaaggtg 1621 cagggcctga gctacctgct gcggagaaag aacggcgcca agcaccccgt gtaccccaca 1681 gcccctgccg tggcctttcc agccggcagc cagaacaacg ccttcatcga gttcatggaa 1741 tccgccttcg tgaactacgt ggacatcagc cacctggtca tccacgaggt ggcccacttc 1801 atctgggtca acaccgtgtc caaagaactg aaagagaaat ggatccagat cgggcagtgg 1861 tacaaagagc ccctgagccc cagcgaatgg gccacaaagc tggaagtgga attcgtgtcc 1921 gcctacgccc atgataagaa ccccgccgag gacttcgccg agagcatggc cacctacgtg 1981 ctgaacagca agctgctgaa ctccagaagc ttcgacaagt tcaagtggat tcaggacaat 2041 ctgttcggcg gaggcttcta catcaccacc ggcacccaca agttcgacgt gatcaacctg 2101 ggcaatgagg tgtactactt ccccggcaaa gtgaccagag tgcgggccaa ggtgctgggc 2161 tcccctaccg aggacaagct ggtcaagatc tacatcagcc tgctgtccag cgacggcagc 2221 gagggctgtg ccaagcacgg ctacgcccgg atcttcagcg agcagcagac cttccgggac 2281 ctgtacttcc acaccgagga tagaagcccc tgcagccaca agctgtacgg cgagttcacc 2341 atgaacaagc acgagagcag aggccggtgg accgccgagt ccatgatctt caccggcgag 2401 aacaacatcg agagatacgt gggcctgggc tctttccact tctacctgta cgtgaacaac 2461 cagaacgagg acgtggaaaa gcctatccct ctgctggact ccatcagcat ctacacacac 2521 aacgccgccg agactaacga cgctctgctg cggctgcacg tgatggtgct ggaaaatgaa 2581 ctgatcaaag agcacggcgg accttacgcc agcttcgccg cccacgagaa caaggcctac 2641 tcctacgaga gccggaccta caagatgtac cctcccgagt tcaacaccct gatgctgaag 2701 gccgactact tcatcagaga tatcaacacc agaggcttcc gcgaagtgaa catggacagc 2761 tgcaagagct acaccaatat ggacacccgg aacctgaagt gcttccaggt gctgaacccc 2821 gtgaccatcc ccaagtactg tatcggctcc acctactttc tgagacaggt gtccatcgag 2881 gatatcgccg gcaatctgga aaccgtgaat atcgccagcg acaagtacag cgccagactg 2941 caccctatcg gcgtgcggga caagcagaaa cccgtggtgt ccaacgtgcg ggtgtcctcc 3001 aagcccgcca acgagtacca cgacggcgag acaatcgtgt ccctgagctt caacgtgcac 3061 gacaacctgg ccggcgtgta ctacatcttc gtgtacctgc gggatcccca cggcggaaag 3121 cacagaagcg acatcgaccg ggccagcctg cctaccggca cagagaacaa gcagatcaat 3181 cacaagatcc tgctgcccaa gggctccatg ggcggcacct ggatgctgga agagatcaag 3241 gccgtggact cctgcaagaa cgaggcccgg aatatctaca cccactccgt gtacgtgcag 3301 aacgacggcg cgccgtcgac ctccatcacg gcctataaga gtgaggggga gtcagcggag 3361 ttctccttcc cactcaacct tggagaggaa agcctgcagg gagagttgag atggaaggca 3421 gagaaggctc cttcttccca gtcctggatc accttctccc taaagaacca aaaggtgtct 3481 gtgcagaagt ctactagcaa ccccaagttc cagctgtccg aaacgctccc actcaccctt 3541 cagatacccc aggtctccct tcagtttgct ggttctggca acctgaccct gactctggac 3601 agagggatac tgtatcagga agtgaacctg gtggtgatga aagtgactca gcccgacagc 3661 aacactttga cctgtgaggt gatgggaccc acctcaccca agatgagact gatcttgaag 3721 caggagaatc aggaggccag ggtctccagg caggagaaag tgattcaagt gcaggcccct 3781 gaagcagggg tgtggcaatg tctactgagt gaaggtgaag aggtcaagat ggactccaag 3841 atccaggttt tatccaaagg gttgaattcc ggcggcggct ccggaggcgg taccggtggc 3901 ggcgacctag ccccacagat gcttcgagaa ctccaggaga ctaatgcggc gctgcaagac 3961 gtgagagagc tcttgcgaca gcaggtcaag gagatcacct tcctgaagaa tacggtgatg 4021 gaatgtgacg cctgcggtct agacagaaac ctgcctccac tagcacctct gggcccacac 4081 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 4141 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 4201 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 4261 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 4321 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 4381 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 4441 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 4501 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 4561 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 4621 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 4681 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 4741 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 4801 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 4861 cattggggcc ctgactacaa agaccatgac ggtgattata aagatcatga catcgattac 4921 aaggatgacg atgacaaggg ccctcaccac caccatcacc actaattaat taaggatccc 4981 ccgacctcga cctctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat 5041 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttggt cgagatccct 5101 cggagatctc tagctagagg atcgatcccc gccccggacg aactaaacct gactacgaca 5161 tctctgcccc ttcttcgcgg ggcagtgcat gtaatccctt cagttggttg gtacaacttg 5221 ccaactgggc cctgttccac atgtgacacg gggggggacc aaacacaaag gggttctctg 5281 actgtagttg acatccttat aaatggatgt gcacatttgc caacactgag tggctttcat 5341 cctggagcag actttgcagt ctgtggactg caacacaaca ttgcctttat gtgtaactct 5401 tggctgaagc tcttacacca atgctggggg acatgtacct cccaggggcc caggaagact 5461 acgggaggct acaccaacgt caatcagagg ggcctgtgta gctaccgata agcggaccct 5521 caagagggca ttagcaatag tgtttataag gcccccttgt taaccctaaa cgggtagcat 5581 atgcttcccg ggtagtagta tatactatcc agactaaccc taattcaata gcatatgtta 5641 cccaacggga agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga 5701 tatctcccac cccatgagct gtcacggttt tatttacatg gggtcaggat tccacgaggg 5761 tagtgaacca ttttagtcac aagggcagtg gctgaagatc aaggagcggg cagtgaactc 5821 tcctgaatct tcgcctgctt cttcattctc cttcgtttag ctaatagaat aactgctgag 5881 ttgtgaacag taaggtgtat gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag 5941 aataaaattt ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc 6001 acaaacccct tgggcaataa atactagtgt aggaatgaaa cattctgaat atctttaaca 6061 atagaaatcc atggggtggg gacaagccgt aaagactgga tgtccatctc acacgaattt 6121 atggctatgg gcaacacata atcctagtgc aatatgatac tggggttatt aagatgtgtc 6181 ccaggcaggg accaagacag gtgaaccatg ttgttacact ctatttgtaa caaggggaaa 6241 gagagtggac gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa 6301 acggggctcc acgccaatgg ggcccataaa caaagacaag tggccactct tttttttgaa 6361 attgtggagt gggggcacgc gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa 6421 ataagggtgt aataacttgg ctgattgtaa ccccgctaac cactgcggtc aaaccacttg 6481 cccacaaaac cactaatggc accccgggga atacctgcat aagtaggtgg gcgggccaag 6541 ataggggcgc gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca 6601 agcacagggt tgttggtcct catattcacg aggtcgctga gagcacggtg ggctaatgtt 6661 gccatgggta gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc 6721 tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc 6781 tgggtagtat atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc 6841 tgggtagcat atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc 6901 cgggtagcat atgctatcct aatagagatt agggtagtat atgctatcct aatttatatc 6961 tgggtagcat atactaccca aatatctgga tagcatatgc tatcctaatc tatatctggg 7021 tagcatatgc tatcctaatc tatatctggg tagcataggc tatcctaatc tatatctggg 7081 tagcatatgc tatcctaatc tatatctggg tagtatatgc tatcctaatt tatatctggg 7141 tagcataggc tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg 7201 tagtatatgc tatcctaatc tgtatccggg tagcatatgc tatcctcatg ataagctgtc 7261 aaacatgaga attttcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta 7321 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 7381 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 7441 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 7501 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 7561 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 7621 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 7681 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag 7741 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 7801 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 7861 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 7921 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 7981 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa 8041 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 8101 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 8161 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 8221 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 8281 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 8341 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 8401 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 8461 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 8521 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 8581 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 8641 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 8701 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 8761 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 8821 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 8881 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 8941 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 9001 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 9061 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 9121 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 9181 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 9241 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 9301 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 9361 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 9421 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 9481 ttcacacagg aaacagctat gaccatgatt acgccaagct ctagctagag gtcgaccaat 9541 tctcatgttt gacagcttat catcgcagat ccgggcaacg ttgttgccat tgctgcaggc 9601 gcagaactgg taggtatgga agatctatac attgaatcaa tattggcaat tagccatatt 9661 agtcattggt tatatagcat aaatcaatat tggctattgg ccattgcata cgttgtatct 9721 atatcataat atgtacattt atattggctc atgtccaata tgaccgccat gttgacattg 9781 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 9841 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 9901 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 9961 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 10021 tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 10081 tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat 10141 cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga 10201 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 10261 aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg 10321 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcct 10381 cactctcttc cgcatcgctg tctgcgaggg ccagctgttg ggctcgcggt tgaggacaaa 10441 ctcttcgcgg tctttccagt actcttggat cggaaacccg tcggcctccg aacggtactc 10501 cgccaccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacct //