LOCUS Exported 8291 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 PF3D7_1351800.1-COMP-blac-flag-his SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8291) TITLE Pentamerised full-length protein ectodomains with no N-linked glycans REFERENCE 2 (bases 1 to 8291) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8291 /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 1825..2610 /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 2617..2682 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2689..2706 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 2742..2797 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(2796..2815) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(5033..5051) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5119..5223 /gene="bla" /label=AmpR promoter CDS 5224..6084 /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 6255..6843 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6744..6763 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6997..7014 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7131..7152 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7167..7197 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7205..7221 /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 7210..7232 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7229..7245 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7229..7245 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 7516..7895 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7896..8099 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8050..8070 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 8096..8120 /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 ggccgccgtg 421 aaagagcgga acaacggctt ccccaagcgg ggcgagggca gatacctgtt cggccggaac 481 cagatgaagt cccaggacat cgagaagaac aacaaagaac tgagcaacaa gatggacacc 541 caggacagca agcagtacat cacctacctg taccacagca gcatctgcca gtactgcagc 601 aaagtgacct ccatgctgga aaacaacgac aacgtcgaga tcatcaagtt caaagaaaac 661 aacaagatcg aggacttcgg caagttcacc aagcccatcg tggtgctgct gaagaacatc 721 aacaaagaga acagcctcga gcggagcatc ttctacgagg aactgaagcg gaagggcaag 781 aaggtgcagg tccccgccct ggaagtgaac aacatcatcc tgttcgagag cgacgagatt 841 attaagttct acaagaagct gctgcagaaa gtctccaacg acgacaagag cagcctgcag 901 aaccggggca acatcaagaa cgaccagaag aaaaacaacg ccgactacga caacgactac 961 gataatgacg acaacaacga tgacaatgac gatgacgacg ataacaacta taacaacaac 1021 aatgatgatg gctacgacta ccacaccagc ggcgcgccgt cgacctccat cacggcctat 1081 aagagtgagg gggagtcagc ggagttctcc ttcccactca accttggaga ggaaagcctg 1141 cagggagagt tgagatggaa ggcagagaag gctccttctt cccagtcctg gatcaccttc 1201 tccctaaaga accaaaaggt gtctgtgcag aagtctacta gcaaccccaa gttccagctg 1261 tccgaaacgc tcccactcac ccttcagata ccccaggtct cccttcagtt tgctggttct 1321 ggcaacctga ccctgactct ggacagaggg atactgtatc aggaagtgaa cctggtggtg 1381 atgaaagtga ctcagcccga cagcaacact ttgacctgtg aggtgatggg acccacctca 1441 cccaagatga gactgatctt gaagcaggag aatcaggagg ccagggtctc caggcaggag 1501 aaagtgattc aagtgcaggc ccctgaagca ggggtgtggc aatgtctact gagtgaaggt 1561 gaagaggtca agatggactc caagatccag gttttatcca aagggttgaa ttccggcggc 1621 ggctccggag gcggtaccgg tggcggcgac ctagccccac agatgcttcg agaactccag 1681 gagactaatg cggcgctgca agacgtgaga gagctcttgc gacagcaggt caaggagatc 1741 accttcctga agaatacggt gatggaatgt gacgcctgcg gtctagacag aaacctgcct 1801 ccactagcac ctctgggccc acacccagaa acgctggtga aagtaaaaga tgctgaagat 1861 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 1921 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 1981 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 2041 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2101 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2161 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 2221 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 2281 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 2341 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 2401 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 2461 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 2521 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 2581 gagataggtg cctcactgat taagcattgg ggccctgact acaaagacca tgacggtgat 2641 tataaagatc atgacatcga ttacaaggat gacgatgaca agggccctca ccaccaccat 2701 caccactaat aataaggatc ccccgacctc gacctctggc taataaagga aatttatttt 2761 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc 2821 aaatcatttg gtcgagatcc ctcggagatc tctagctaga ggatcgatcc ccgccccgga 2881 cgaactaaac ctgactacga catctctgcc ccttcttcgc ggggcagtgc atgtaatccc 2941 ttcagttggt tggtacaact tgccaactgg gccctgttcc acatgtgaca cgggggggga 3001 ccaaacacaa aggggttctc tgactgtagt tgacatcctt ataaatggat gtgcacattt 3061 gccaacactg agtggctttc atcctggagc agactttgca gtctgtggac tgcaacacaa 3121 cattgccttt atgtgtaact cttggctgaa gctcttacac caatgctggg ggacatgtac 3181 ctcccagggg cccaggaaga ctacgggagg ctacaccaac gtcaatcaga ggggcctgtg 3241 tagctaccga taagcggacc ctcaagaggg cattagcaat agtgtttata aggccccctt 3301 gttaacccta aacgggtagc atatgcttcc cgggtagtag tatatactat ccagactaac 3361 cctaattcaa tagcatatgt tacccaacgg gaagcatatg ctatcgaatt agggttagta 3421 aaagggtcct aaggaacagc gatatctccc accccatgag ctgtcacggt tttatttaca 3481 tggggtcagg attccacgag ggtagtgaac cattttagtc acaagggcag tggctgaaga 3541 tcaaggagcg ggcagtgaac tctcctgaat cttcgcctgc ttcttcattc tccttcgttt 3601 agctaataga ataactgctg agttgtgaac agtaaggtgt atgtgaggtg ctcgaaaaca 3661 aggtttcagg tgacgccccc agaataaaat ttggacgggg ggttcagtgg tggcattgtg 3721 ctatgacacc aatataaccc tcacaaaccc cttgggcaat aaatactagt gtaggaatga 3781 aacattctga atatctttaa caatagaaat ccatggggtg gggacaagcc gtaaagactg 3841 gatgtccatc tcacacgaat ttatggctat gggcaacaca taatcctagt gcaatatgat 3901 actggggtta ttaagatgtg tcccaggcag ggaccaagac aggtgaacca tgttgttaca 3961 ctctatttgt aacaagggga aagagagtgg acgccgacag cagcggactc cactggttgt 4021 ctctaacacc cccgaaaatt aaacggggct ccacgccaat ggggcccata aacaaagaca 4081 agtggccact cttttttttg aaattgtgga gtgggggcac gcgtcagccc ccacacgccg 4141 ccctgcggtt ttggactgta aaataagggt gtaataactt ggctgattgt aaccccgcta 4201 accactgcgg tcaaaccact tgcccacaaa accactaatg gcaccccggg gaatacctgc 4261 ataagtaggt gggcgggcca agataggggc gcgattgctg cgatctggag gacaaattac 4321 acacacttgc gcctgagcgc caagcacagg gttgttggtc ctcatattca cgaggtcgct 4381 gagagcacgg tgggctaatg ttgccatggg tagcatatac tacccaaata tctggatagc 4441 atatgctatc ctaatctata tctgggtagc ataggctatc ctaatctata tctgggtagc 4501 atatgctatc ctaatctata tctgggtagt atatgctatc ctaatttata tctgggtagc 4561 ataggctatc ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt 4621 atatgctatc ctaatctgta tccgggtagc atatgctatc ctaatagaga ttagggtagt 4681 atatgctatc ctaatttata tctgggtagc atatactacc caaatatctg gatagcatat 4741 gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagcatag 4801 gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat 4861 gctatcctaa tttatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat 4921 gctatcctaa tctatatctg ggtagtatat gctatcctaa tctgtatccg ggtagcatat 4981 gctatcctca tgataagctg tcaaacatga gaattttctt gaagacgaaa gggcctcgtg 5041 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 5101 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 5161 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 5221 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 5281 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 5341 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 5401 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 5461 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 5521 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5581 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5641 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5701 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5761 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5821 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5881 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 5941 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 6001 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 6061 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 6121 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 6181 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6241 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6301 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 6361 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 6421 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6481 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6541 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6601 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6661 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6721 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6781 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6841 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6901 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 6961 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 7021 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 7081 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 7141 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 7201 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 7261 ctctagctag aggtcgacca attctcatgt ttgacagctt atcatcgcag atccgggcaa 7321 cgttgttgcc attgctgcag gcgcagaact ggtaggtatg gaagatctat acattgaatc 7381 aatattggca attagccata ttagtcattg gttatatagc ataaatcaat attggctatt 7441 ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc tcatgtccaa 7501 tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt 7561 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 7621 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 7681 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 7741 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg 7801 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc 7861 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca 7921 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 7981 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg 8041 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 8101 gtttagtgaa ccgtcagatc ctcactctct tccgcatcgc tgtctgcgag ggccagctgt 8161 tgggctcgcg gttgaggaca aactcttcgc ggtctttcca gtactcttgg atcggaaacc 8221 cgtcggcctc cgaacggtac tccgccaccg agggacctga gcgagtccgc atcgaccgga 8281 tcggaaaacc t //