LOCUS Exported 9745 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 CPN60-COMP-blac-flag-his SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9745) TITLE Pentamerised full-length protein ectodomains with no N-linked glycans REFERENCE 2 (bases 1 to 9745) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9745 /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)" protein_bind 2389..2400 /label=ZF binding site /bound_moiety="CCR5TC zinc-finger domain" /note="target sequence for the CCR5TC zinc-finger domain from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 2008; Gross et al., 2013)" CDS 3277..4062 /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 4069..4134 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4141..4158 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 4196..4251 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(4250..4269) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(6487..6505) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6573..6677 /gene="bla" /label=AmpR promoter CDS 6678..7538 /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 7709..8297 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8198..8217 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8451..8468 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8585..8606 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8621..8651 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8659..8675 /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 8664..8686 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8683..8699 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8683..8699 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 8970..9349 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 9350..9553 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 9504..9524 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 9550..9574 /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 ggccgccatc 421 aagaagaagc ggagccccaa caacaagaac cggctgttca tcaacaagcg gctgaagtac 481 atcaacagca agatcatcag ccggcggaaa gaaaactacg tgaagatgaa gatgaccgag 541 aacaaagtga agggcaagga catcatctac ggcaacgagt gccggaacga gctgctgaag 601 ggcatcctga ccgtgtccga cgtggtcaag ctgaccctgg gccccagagg cagaaacgtg 661 ctgctggaaa aagagtacgg cagccctctg atcatcaacg acggcgtgac aatcgccaag 721 aatatcgccc tgaaggaccg gaagaaaaac aacggcgtga agctgatgca ggaaagcacc 781 aatatcgcca acgacaaggc cggcgacggc accagctcta ccgccctgat gaccgccacc 841 atcaccaaga agggaatcga gcaggtcaac cggaaccaca accccatccc catccagcgg 901 ggcatccagc tggcctccaa gatgatcatt gagaagatca agagcctgag cacccctatc 961 aagacctata aggacatcct gaacattgcc accattgcca gcaacaacga cgtgcacatg 1021 ggccagatca ttgccaacgc ctacgacaag ctgggcaaga acgccgccat tatcctggac 1081 gacaacgccg acatcaacga taagctggaa ttcaccgagg gctacaactt cgaccggggc 1141 atcatcaacc cctacctgct gtacaacgag aacaaggact acatcgagta cagcaacgtg 1201 gccaccctga tcaccgacca gaacatcgac aacatccaga gcatcctgcc catcctggaa 1261 atctttgcca agaacaagca gcccctgtgc atcattgccg acgacttcag caacgaggtg 1321 ctgcagaccc tgattattaa caagctgaaa ggggccatca aggtggtgcc catcagagcc 1381 cctagcttcg gcgaccggcg gaaggactac ctgaaggatc tgtgcatcgt gaccaactcc 1441 aagtacatct ccgccgacgt gggcctggac ctgaacaacc tgcacaacaa catggccagc 1501 ttcgacaaca actacctgag cctgctgggc agcgccaata ccctgatcgt gaagaaggac 1561 agaaccagcc tgattaccaa agaagagtac aagaaagaga tcgacgagcg gatcaacgtc 1621 ctgaagaaag agtatgagga aaccacctct aagtacgaca aagagaagct gaacgagaga 1681 atcgccgccc tgagcggcgg cattgccaag atcctgatcg gcggaaacag cgagacagag 1741 cagaaagagc ggaagtttaa gtacgaggac gccaccaacg ccgtgaagtc cgccatcgac 1801 atcggctacg tgccaggcgg cggagtgacc tatctggaaa tcatcaagag caacttcatc 1861 caggaaatcc acaagaagat cgaggaagat ctccagatca gctccaacaa cgatgagaag 1921 aagtacctcg agctgatcgg caacctggaa agcgagatgg aactgcagaa gatgggcgcc 1981 aacatcgtgg tgtccagcct ggacgtgatc accaagcaga tcgccgacaa cgctggcgtg 2041 aacggcgaca atgtcgtgaa gatcatcctg aatagcaagg ataagtacgg cttcggctac 2101 gacgtcaaca ccaacaagtt cgtgaacatg gtcgagaagg ggatcatcga cagcaccaac 2161 gtgatcatct ccgtgatcaa gaacagctgc tctatcgcca gcatggtgct gaccaccgag 2221 tgcatgatgg tggaccacga gaagaaggat aagggaatcc tggacagcag catcaactcc 2281 ccaaactacc tgtccaagca ccggcggacc tacaagcaca agctgcacga cgacgaggac 2341 accgacgagg atgacgaaga ggacgaggac gatgaggacg acgaagatga tgaggatgac 2401 gacgactacg atgacgaaga tgaagaggac gaagaggatg aagaggacga ggatgacgag 2461 gacgatgagg atagcatgaa cgacggctac aattacgacg aaggcgcgcc gtcgacctcc 2521 atcacggcct ataagagtga gggggagtca gcggagttct ccttcccact caaccttgga 2581 gaggaaagcc tgcagggaga gttgagatgg aaggcagaga aggctccttc ttcccagtcc 2641 tggatcacct tctccctaaa gaaccaaaag gtgtctgtgc agaagtctac tagcaacccc 2701 aagttccagc tgtccgaaac gctcccactc acccttcaga taccccaggt ctcccttcag 2761 tttgctggtt ctggcaacct gaccctgact ctggacagag ggatactgta tcaggaagtg 2821 aacctggtgg tgatgaaagt gactcagccc gacagcaaca ctttgacctg tgaggtgatg 2881 ggacccacct cacccaagat gagactgatc ttgaagcagg agaatcagga ggccagggtc 2941 tccaggcagg agaaagtgat tcaagtgcag gcccctgaag caggggtgtg gcaatgtcta 3001 ctgagtgaag gtgaagaggt caagatggac tccaagatcc aggttttatc caaagggttg 3061 aattccggcg gcggctccgg aggcggtacc ggtggcggcg acctagcccc acagatgctt 3121 cgagaactcc aggagactaa tgcggcgctg caagacgtga gagagctctt gcgacagcag 3181 gtcaaggaga tcaccttcct gaagaatacg gtgatggaat gtgacgcctg cggtctagac 3241 agaaacctgc ctccactagc acctctgggc ccacacccag aaacgctggt gaaagtaaaa 3301 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 3361 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 3421 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 3481 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 3541 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 3601 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 3661 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 3721 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 3781 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 3841 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 3901 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 3961 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 4021 agacagatcg ctgagatagg tgcctcactg attaagcatt ggggccctga ctacaaagac 4081 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caagggccct 4141 caccaccacc atcaccacta attaattaag gatcccccga cctcgacctc tggctaataa 4201 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg 4261 acatatggga gggcaaatca tttggtcgag atccctcgga gatctctagc tagaggatcg 4321 atccccgccc cggacgaact aaacctgact acgacatctc tgccccttct tcgcggggca 4381 gtgcatgtaa tcccttcagt tggttggtac aacttgccaa ctgggccctg ttccacatgt 4441 gacacggggg gggaccaaac acaaaggggt tctctgactg tagttgacat ccttataaat 4501 ggatgtgcac atttgccaac actgagtggc tttcatcctg gagcagactt tgcagtctgt 4561 ggactgcaac acaacattgc ctttatgtgt aactcttggc tgaagctctt acaccaatgc 4621 tgggggacat gtacctccca ggggcccagg aagactacgg gaggctacac caacgtcaat 4681 cagaggggcc tgtgtagcta ccgataagcg gaccctcaag agggcattag caatagtgtt 4741 tataaggccc ccttgttaac cctaaacggg tagcatatgc ttcccgggta gtagtatata 4801 ctatccagac taaccctaat tcaatagcat atgttaccca acgggaagca tatgctatcg 4861 aattagggtt agtaaaaggg tcctaaggaa cagcgatatc tcccacccca tgagctgtca 4921 cggttttatt tacatggggt caggattcca cgagggtagt gaaccatttt agtcacaagg 4981 gcagtggctg aagatcaagg agcgggcagt gaactctcct gaatcttcgc ctgcttcttc 5041 attctccttc gtttagctaa tagaataact gctgagttgt gaacagtaag gtgtatgtga 5101 ggtgctcgaa aacaaggttt caggtgacgc ccccagaata aaatttggac ggggggttca 5161 gtggtggcat tgtgctatga caccaatata accctcacaa accccttggg caataaatac 5221 tagtgtagga atgaaacatt ctgaatatct ttaacaatag aaatccatgg ggtggggaca 5281 agccgtaaag actggatgtc catctcacac gaatttatgg ctatgggcaa cacataatcc 5341 tagtgcaata tgatactggg gttattaaga tgtgtcccag gcagggacca agacaggtga 5401 accatgttgt tacactctat ttgtaacaag gggaaagaga gtggacgccg acagcagcgg 5461 actccactgg ttgtctctaa cacccccgaa aattaaacgg ggctccacgc caatggggcc 5521 cataaacaaa gacaagtggc cactcttttt tttgaaattg tggagtgggg gcacgcgtca 5581 gcccccacac gccgccctgc ggttttggac tgtaaaataa gggtgtaata acttggctga 5641 ttgtaacccc gctaaccact gcggtcaaac cacttgccca caaaaccact aatggcaccc 5701 cggggaatac ctgcataagt aggtgggcgg gccaagatag gggcgcgatt gctgcgatct 5761 ggaggacaaa ttacacacac ttgcgcctga gcgccaagca cagggttgtt ggtcctcata 5821 ttcacgaggt cgctgagagc acggtgggct aatgttgcca tgggtagcat atactaccca 5881 aatatctgga tagcatatgc tatcctaatc tatatctggg tagcataggc tatcctaatc 5941 tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc tatcctaatt 6001 tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc tatcctaatc 6061 tatatctggg tagtatatgc tatcctaatc tgtatccggg tagcatatgc tatcctaata 6121 gagattaggg tagtatatgc tatcctaatt tatatctggg tagcatatac tacccaaata 6181 tctggatagc atatgctatc ctaatctata tctgggtagc atatgctatc ctaatctata 6241 tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc ctaatctata 6301 tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc ctaatctata 6361 tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc ctaatctgta 6421 tccgggtagc atatgctatc ctcatgataa gctgtcaaac atgagaattt tcttgaagac 6481 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 6541 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 6601 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 6661 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 6721 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 6781 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 6841 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6901 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact 6961 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 7021 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 7081 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 7141 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 7201 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg 7261 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 7321 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 7381 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 7441 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 7501 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 7561 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 7621 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 7681 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 7741 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 7801 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 7861 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7921 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7981 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 8041 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 8101 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 8161 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 8221 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 8281 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 8341 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 8401 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 8461 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 8521 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 8581 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 8641 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 8701 atgattacgc caagctctag ctagaggtcg accaattctc atgtttgaca gcttatcatc 8761 gcagatccgg gcaacgttgt tgccattgct gcaggcgcag aactggtagg tatggaagat 8821 ctatacattg aatcaatatt ggcaattagc catattagtc attggttata tagcataaat 8881 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat 8941 tggctcatgt ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta 9001 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 9061 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac 9121 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt 9181 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat 9241 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga 9301 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 9361 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 9421 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 9481 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 9541 tataagcaga gctcgtttag tgaaccgtca gatcctcact ctcttccgca tcgctgtctg 9601 cgagggccag ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc 9661 ttggatcgga aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt 9721 ccgcatcgac cggatcggaa aacct //