LOCUS Exported 6625 bp ds-DNA circular SYN 05-AUG-2022 DEFINITION Mammalian expression of cell-surface protein extracellular domain fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells.. ACCESSION . VERSION . KEYWORDS pD649-HAsp-CD40-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6625) 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 6625) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 5, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6625 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 194..214 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" polyA_signal 332..443 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 432..449 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS complement(589..606) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(607..651) /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" primer_bind complement(1987..2006) /label=pMT2-F /note="Synthetic intron, forward primer" promoter complement(2019..2037) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2020..2037) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2247..2271) /label=LNCX /note="Human CMV promoter, forward primer" promoter complement(2268..2471) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(2297..2317) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 2472..2851 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" protein_bind 3018..3034 /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 complement(3490..3507) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3490..3506) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3605..3622) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3776..4364) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3856..3875) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(4450..4469) /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind complement(4475..4494) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter 4536..4627 /gene="bla" /label=AmpR promoter CDS 4628..5488 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4846..4865) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" polyA_signal 5541..5662 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5552..5571) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5606..5625 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(5792..6391) /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5875..5895) /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind 6372..6391 /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" promoter complement(6410..6457) /label=EM7 promoter /note="synthetic bacterial promoter " promoter complement(6505..209) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6519..29 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(6573..6592) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" ORIGIN 1 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct 61 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca 121 cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct 181 ggggactttc cacaccctaa ctgacacaca ttccacagaa ttaattcgcg ttaaattttt 241 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 301 aagaatagac cgagataggg ttgagtgttg tgatgcaatt tcctcatttt attaggaaag 361 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga 421 tggctggcaa ctagaaggca cagtcgaggc tgattttctc tatctattta ttttactact 481 caagactatc ttgcaaagta gaagaaccct gtctgcagta tgacgaccgt gtatacccga 541 ttggcttacc gggtaacgcg ttgtgccagt gactacaggc cggccttagt ggtgatggtg 601 gtggtgttca tgccactcaa tcttctgagc ctcgaaaata tcattaagac cgctgccgcc 661 ggggctcagg ctcagggact tctgggtgta gtggttgtgc agggcctcgt gcatcacgct 721 gcagctgaac acgttgccct gctgccacct ggacttgtcc acggtcagct tgctgtacag 781 gaagaagctg ccgtcgctgt ccagcactgg gggggtggtc ttgtagttgt tctcgggctg 841 gccgttgctc tcccactcca cggcgatatc gctggggtag aagcccttca ccagacaggt 901 cagggacacc tggttcttgg tcatctcctc ccggctgggg ggcagggtgt acacctgggg 961 ctcccgtggc tggcccttgg ccttgctgat tgtcttttcg attggggcag ccagggcctt 1021 gttggagact ttgcacttgt attctttgcc gttcagccag tcctggtgca gcacggtcag 1081 cacggacacc accctgtagg tgctgttgta ctgctcctct ctgggcttgg tcttggcgtt 1141 gtgcacctcc acgccgtcca cgtaccagtt gaacttcacc tctgggtcct cgtgggacac 1201 ggccaccacc acgcaggtca cctcgggggt cctgctgatc atcagggtgt ccttgggctt 1261 gggggggaac aggaacacgg aaggccctcc cagcagttct ggagctgggc agggggggca 1321 ggtgtgggtc ttgtcgccgc ccggcgcgcc tctaagccgg tcctgagggc cgcacacaac 1381 atctgtcttg ttggtgccgg cctgctgcac aaccagatcc tttgtctcgc agctggtcca 1441 agggtgacac ttctcgaagg cgctggacac attgctgaag aagcccacag gacaaggctc 1501 gcagatggta tcggacacgc ctgtggcaat ctgcttcact ccgaagccag ggctacagct 1561 tctgtgcagc acacagcttt cacaggcctc gctggtacag tgccatcctt cctcgcatgt 1621 acagatggtg tctgtctcgc tggtgccctt ctgctgcact ctcagtccca gattggggtc 1681 gcagtacttg tgctggtggc agtgtgtctc tctattccag gtgtccagga actcgctctc 1741 gccgcaaggc agacactctg tctcggtgaa ctcggtgcaa tcggacacca gtttctgtcc 1801 aggctggcac aggctgcagc actggctgtt gatcaggtac tgcttctctc tacaggctgt 1861 aggaggttcg gcggccgcag cgaacaccag gcagaagatg tagctcaggg caatgatggt 1921 cttcatggtg gcgctagcag gcgcgttttc gatagaaccg aggtgcagtt ggacctggga 1981 gtggacacct gtggagagaa aggcaaagtg gatgttattc tatagtgtca cctaaatcgt 2041 atgtgtatga tacataaggt tatgtattaa ttgtagccgc gttctaacga agccaagggg 2101 gtgggcctat agactctata ggcggtactt acgtcactct tggcacgggg aatccgcgtt 2161 ccaatgcacc gttcccggcc gcggaggctg gatcggtccc ggtgtcttct atggaggtca 2221 aaacagcgtg gatggcgtct ccaggcgatc tgacggttca ctaaacgagc tctgcttata 2281 tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga 2341 cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg 2401 tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa 2461 aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag 2521 tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca 2581 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt 2641 aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac 2701 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc 2761 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat 2821 tactattaat aactagtcaa taatcaatgc caacatggcg gtcatattgg acatgagcca 2881 atataaatgt acatattatg atatagatac aacgtatgca atggccaata gccaatattg 2941 atttatgcta tataaccaat gaataatatg gctaatggcc aatattgagt caacgccact 3001 gtgcatgaag ctcgtaattg ttatccgctc acaattaagg gcgacacaaa atttattcta 3061 aatgataata aatactgata acatcttata gtttgtatta tattttgtat tatcgttgac 3121 atgtataatt ttgatatcaa aaactgattt tccctttatt attttcgaga tttattttct 3181 taattctctt taacaaacta gaaatattgt atatacaaaa aatcataaat aatagatgaa 3241 tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt atcttattta aagtgcgttg 3301 cttttttctc atttataagg ttaaataatt ctcatatatc aagcaaagtg acaggcgccc 3361 ttaaatattc tgacaaatgc tctttcccta aactcccccc ataaaaaaac ccgccgaagc 3421 gggtttttac gttatttgcg gattaacgat tactcgttat cagaaccgcc cagggggccc 3481 gagcttaaga ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac gcaaaaaggc 3541 catccgtcag gggccttctg cttagtttga tgcctggcag ttccctactc tcgccttccg 3601 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 3661 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 3721 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 3781 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 3841 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 3901 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 3961 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 4021 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 4081 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 4141 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tgggctaact 4201 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 4261 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 4321 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4381 tttctacggg gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt aagggatttt 4441 ggtcatgagc aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt 4501 cagaattggt taattggttg taacactgac ccctatttgt ttatttttct aaatacattc 4561 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 4621 gaagaatatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 4681 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 4741 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 4801 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 4861 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 4921 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 4981 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 5041 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 5101 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 5161 cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 5221 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 5281 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatccggag ccggtgagcg 5341 tggttctcgc ggtatcatcg cagcgctggg gccagatggt aagccctccc gtatcgtagt 5401 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 5461 aggtgcctca ctgattaagc attggtaatg accatgatta cgccaagctc tagctagagg 5521 tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca caactagaat 5581 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 5641 tataagctgc aataaacaag ttggggtggg cgaagaactc cagcatgaga tccccgcgct 5701 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg 5761 cggcggtgga atcgaaatct cgtagcacgt gtcaggcacc gggcttgcgg gtcatgcacc 5821 aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt 5881 agaaggggag gttgcggggc gcggatgtct ccaggaaggc gggcaccccg gcgcgctcgg 5941 ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg 6001 agacgccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc 6061 cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga 6121 tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg 6181 cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt 6241 cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc gacggtgtgg cgcgtggcgg 6301 ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct ggggacgtcg tcgcgggtgg 6361 cgaggcgcac cgtgggcttg tactcggtca tggtttagtt cctcaccttg tcgtattata 6421 ctatgccgat atactatgcc gatgattaat tgtcaacacg tgctgatcag atccgaaaat 6481 ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa agcctcctca 6541 ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat 6601 tagtcagcca tggggcggag aatgg //