LOCUS Exported 7080 bp ds-DNA circular SYN 06-JAN-2023 DEFINITION Mammalian cell surface expression of His- and FLAG-tagged full-length protein for binding and signaling assays. ACCESSION . VERSION . KEYWORDS pTT3-SP-6xHis-KIR2DL1(FL)-FLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7080) AUTHORS Wojtowicz WM, Vielmetter J, Fernandes RA, Siepe DH, Eastman CL, Chisholm GB, Cox S, Klock H, Anderson PW, Rue SM, Miller JJ, Glaser SM, Bragstad ML, Vance J, Lam AW, Lesley SA, Zinn K, Garcia KC TITLE A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions. JOURNAL Cell. 2020 Aug 20;182(4):1027-1043.e17. doi: 10.1016/j.cell.2020.07.025. PUBMED 32822567 REFERENCE 2 (bases 1 to 7080) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 6, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7080 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 90..110 /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" CDS 1095..1118 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" polyA_signal 1175..1230 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(1229..1248) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(3466..3484) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3552..3656 /gene="bla" /label=AmpR promoter CDS 3657..4517 /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" primer_bind complement(3875..3894) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4688..5276 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5177..5196 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5430..5447 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5564..5585 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5600..5630 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5638..5654 /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 5643..5665 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5662..5678 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5662..5678 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 5949..6328 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6329..6532 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6483..6503 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 6529..6553 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind 7034..7053 /label=pMT2-F /note="Synthetic intron, forward primer" ORIGIN 1 gcggccgcgc tagcgccacc atgtctctgc tggttgtttc tatggcctgc gtgggattct 61 tcctgctgca aggcgcttgg cctggatctc accaccatca ccatcatcac gagggcgtgc 121 acagaaagcc tagcctgctt gctcatcccg gacctctggt caagagcgaa gagacagtga 181 tcctgcagtg ttggagcgac gtgatgttcg agcactttct gctgcaccgc gagggcatgt 241 tcaacgacac actgagactg atcggcgagc accacgatgg cgtgtccaag gccaatttca 301 gcatcagcag aatgacccag gacctggccg gcacctacag atgttatggc tctgtgacac 361 acagccccta ccaggtgtca gcccctagcg atcctctgga catcgtgatc atcggcctgt 421 acgagaagcc aagcctgtct gcacagcctg gacctacagt gctggccggc gaaaatgtga 481 ccctgagctg tagcagccgg tccagctacg atatgtacca cctgagcaga gagggcgaag 541 cccacgaaag aaggctgcct gccggaccta aagtgaacgg caccttccag gccgattttc 601 cactgggacc tgccacacac ggcggcacat atagatgctt cggcagcttc cacgacagcc 661 cttacgagtg gtctaagagc agcgatccac tgctggtgtc cgtgaccggc aatcctagca 721 atagctggcc ttctccaacc gagcctagca gcaagacagg caaccccaga catctgcaca 781 tcctgatcgg caccagcgtg gtcatcatcc tgttcatcct gctgttcttt ctgctccaca 841 gatggtgcag caacaagaaa aacgccgccg tgatggatca agagagcgcc ggcaatagaa 901 ccgccaacag cgaggattcc gacgagcagg accctcaaga agtgacctac acacagctga 961 accactgcgt gttcacccag cggaagatca ccagacctag ccagaggcct aagacacctc 1021 ctaccgacat catcgtgtac accgagctgc ctaacgccga gagcagatcc aaggtggtgt 1081 cttgccctgg cagcgactac aaggacgacg acgataagta aacgcgttct agacacgtga 1141 ttaattaagg atcccccgac ctcgacctct ggctaataaa ggaaatttat tttcattgca 1201 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat 1261 ttggtcgaga tccctcggag atctctagct agaggatcga tccccgcccc ggacgaacta 1321 aacctgacta cgacatctct gccccttctt cgcggggcag tgcatgtaat cccttcagtt 1381 ggttggtaca acttgccaac tgggccctgt tccacatgtg acacgggggg ggaccaaaca 1441 caaaggggtt ctctgactgt agttgacatc cttataaatg gatgtgcaca tttgccaaca 1501 ctgagtggct ttcatcctgg agcagacttt gcagtctgtg gactgcaaca caacattgcc 1561 tttatgtgta actcttggct gaagctctta caccaatgct gggggacatg tacctcccag 1621 gggcccagga agactacggg aggctacacc aacgtcaatc agaggggcct gtgtagctac 1681 cgataagcgg accctcaaga gggcattagc aatagtgttt ataaggcccc cttgttaacc 1741 ctaaacgggt agcatatgct tcccgggtag tagtatatac tatccagact aaccctaatt 1801 caatagcata tgttacccaa cgggaagcat atgctatcga attagggtta gtaaaagggt 1861 cctaaggaac agcgatatct cccaccccat gagctgtcac ggttttattt acatggggtc 1921 aggattccac gagggtagtg aaccatttta gtcacaaggg cagtggctga agatcaagga 1981 gcgggcagtg aactctcctg aatcttcgcc tgcttcttca ttctccttcg tttagctaat 2041 agaataactg ctgagttgtg aacagtaagg tgtatgtgag gtgctcgaaa acaaggtttc 2101 aggtgacgcc cccagaataa aatttggacg gggggttcag tggtggcatt gtgctatgac 2161 accaatataa ccctcacaaa ccccttgggc aataaatact agtgtaggaa tgaaacattc 2221 tgaatatctt taacaataga aatccatggg gtggggacaa gccgtaaaga ctggatgtcc 2281 atctcacacg aatttatggc tatgggcaac acataatcct agtgcaatat gatactgggg 2341 ttattaagat gtgtcccagg cagggaccaa gacaggtgaa ccatgttgtt acactctatt 2401 tgtaacaagg ggaaagagag tggacgccga cagcagcgga ctccactggt tgtctctaac 2461 acccccgaaa attaaacggg gctccacgcc aatggggccc ataaacaaag acaagtggcc 2521 actctttttt ttgaaattgt ggagtggggg cacgcgtcag cccccacacg ccgccctgcg 2581 gttttggact gtaaaataag ggtgtaataa cttggctgat tgtaaccccg ctaaccactg 2641 cggtcaaacc acttgcccac aaaaccacta atggcacccc ggggaatacc tgcataagta 2701 ggtgggcggg ccaagatagg ggcgcgattg ctgcgatctg gaggacaaat tacacacact 2761 tgcgcctgag cgccaagcac agggttgttg gtcctcatat tcacgaggtc gctgagagca 2821 cggtgggcta atgttgccat gggtagcata tactacccaa atatctggat agcatatgct 2881 atcctaatct atatctgggt agcataggct atcctaatct atatctgggt agcatatgct 2941 atcctaatct atatctgggt agtatatgct atcctaattt atatctgggt agcataggct 3001 atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct 3061 atcctaatct gtatccgggt agcatatgct atcctaatag agattagggt agtatatgct 3121 atcctaattt atatctgggt agcatatact acccaaatat ctggatagca tatgctatcc 3181 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagca taggctatcc 3241 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc 3301 taatttatat ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc 3361 taatctatat ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc 3421 tcatgataag ctgtcaaaca tgagaatttt cttgaagacg aaagggcctc gtgatacgcc 3481 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 3541 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 3601 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 3661 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 3721 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 3781 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 3841 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 3901 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 3961 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 4021 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 4081 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 4141 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 4201 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 4261 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 4321 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 4381 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 4441 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 4501 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 4561 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 4621 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 4681 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 4741 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 4801 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 4861 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 4921 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 4981 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 5041 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 5101 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 5161 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 5221 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 5281 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 5341 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 5401 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 5461 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 5521 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 5581 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 5641 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc 5701 tagaggtcga ccaattctca tgtttgacag cttatcatcg cagatccggg caacgttgtt 5761 gccattgctg caggcgcaga actggtaggt atggaagatc tatacattga atcaatattg 5821 gcaattagcc atattagtca ttggttatat agcataaatc aatattggct attggccatt 5881 gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc caatatgacc 5941 gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 6001 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 6061 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 6121 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 6181 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg 6241 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat 6301 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg 6361 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 6421 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt 6481 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt 6541 gaaccgtcag atcctcactc tcttccgcat cgctgtctgc gagggccagc tgttgggctc 6601 gcggttgagg acaaactctt cgcggtcttt ccagtactct tggatcggaa acccgtcggc 6661 ctccgaacgg tactccgcca ccgagggacc tgagcgagtc cgcatcgacc ggatcggaaa 6721 acctctcgag aaaggcgtct aaccagtcac agtcgcaagg taggctgagc accgtggcgg 6781 gcggcagcgg gtggcggtcg gggttgtttc tggcggaggt gctgctgatg atgtaattaa 6841 agtaggcggt cttgagacgg cggatggtcg aggtgaggtg tggcaggctt gagatccagc 6901 tgttggggtg agtactccct ctcaaaagcg ggcattactt ctgcgctaag attgtcagtt 6961 tccaaaaacg aggaggattt gatattcacc tggcccgatc tggccataca cttgagtgac 7021 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca agtttaaact //