LOCUS Exported 7288 bp ds-DNA circular SYN 13-MAY-2021 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-MR1-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7288) 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 7288) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7288 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 124..143 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 149..168 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 254..842 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 743..762 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 996..1013 /label=L4440 /note="L4440 vector, forward primer" primer_bind 1111..1128 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 1112..1128 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 1584..1600 /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)." enhancer 1767..2146 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2147..2350 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2301..2321 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 2347..2371 /label=LNCX /note="Human CMV promoter, forward primer" promoter 2581..2599 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 2581..2598 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 2612..2631 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 4630..4674 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 4675..4692 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4832..4849) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4838..4949 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5067..5087) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5072..5401 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5252..5387 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5314..5333 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5449..5496 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5515..6114 /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(5515..5534) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6011..6031 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6244..6365 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6281..6300) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6335..6354 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6418..7278) /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 7041..7060 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7279..82) /gene="bla" /label=AmpR promoter ORIGIN 1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 61 tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac 121 attatcgcga gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa 181 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga 241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 361 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 421 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 1021 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc 1081 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg 1141 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct 1201 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg 1261 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa 1321 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc 1381 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga 1441 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc 1501 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag 1561 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt 1621 ggcgttgact caatattggc cattagccat attattcatt ggttatatag cataaatcaa 1681 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg 1741 ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc 1801 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 1861 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 1921 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 1981 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 2041 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 2101 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 2161 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 2221 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 2281 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 2341 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga 2401 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac 2461 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc 2521 cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg 2581 atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc 2641 caggtccaac tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc 2701 atcattgccc tgagctacat cttctgcctg gtgttcgctg cggccgccat tcagagaacc 2761 cctaagatcc aggtgtacag cagacacccc gccgagaacg gcaagagcaa cttcctgaat 2821 tgctacgtgt ccggctttca ccccagcgac atcgaggtgg acctgctgaa gaatggcgag 2881 cggatcgaga aggtggaaca cagcgatctg agcttcagca aggactggtc cttctacctg 2941 ctgtactaca ccgagttcac ccctaccgag aaggacgagt acgcctgcag agtgaaccac 3001 gtgacactga gccagcctaa gatcgtgaag tgggacagag atatgggcgg aggtggaagc 3061 ggaggcggag gatctggtgg cggaggaagt agaacacaca gcctgagata cttccggctg 3121 ggcgtgtccg atcctattca cggcgtgcca gagttcatca gcgtgggcta tgtggactct 3181 caccccatca ccacctacga cagcgtgacc agacagaaag agcccagagc accctggatg 3241 gccgaaaatc tggcccctga tcactgggag agatacaccc aactgctgag aggctggcag 3301 cagatgttca aagtggaact gaagcggctg cagcggcact acaatcacag cggcagccac 3361 acctaccaga gaatgatcgg ctgcgagctg ctggaagatg gcagcacaac aggctttctg 3421 cagtacgcct acgacggcca ggacttcctg atcttcaaca aggataccct gagctggctg 3481 gccgtggata atgtggccca cacaatcaaa caggcctggg aagccaacca gcacgaactg 3541 ctgtaccaga agaactggct ggaagaggaa tgtatcgcct ggctgaagcg gttcctggaa 3601 tacggcaagg acaccctgca gagaacagag cctccactcg tgcgcgtgaa ccggaaagaa 3661 acctttcctg gcgtgacagc cctgttctgc aaggcccatg gcttctaccc acctgagatc 3721 tacatgacct ggatgaagaa cggggaagag atcgtgcaag agatcgacta cggcgacatt 3781 ctgcctagcg gcgacggaac atatcaggct tgggcctcta ttgagctgga ccctcagagc 3841 agcaacctgt actcttgcca cgtggaacac tgcggcgtgc acatggttct gcaggtccca 3901 caagagagcg agacaatccc tctggtcatg ggcgcgccgg gcggcgacaa gacccacacc 3961 tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct gttccccccc 4021 aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggcc 4081 gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 4141 aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg 4201 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa agtctccaac 4261 aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca gccacgggag 4321 ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg 4381 acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga gagcaacggc 4441 cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc 4501 ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt gttcagctgc 4561 agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcccc 4621 ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca tgaacaccac 4681 caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa 4741 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg 4801 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca 4861 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 4921 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt 4981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 5041 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 5101 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 5161 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 5221 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 5281 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 5341 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 5401 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa 5461 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc 5521 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc 5581 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac 5641 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc 5701 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc 5761 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg 5821 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg 5881 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc 5941 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca 6001 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc 6061 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg 6121 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 6181 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 6241 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 6301 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 6361 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta 6421 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 6481 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 6541 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca 6601 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 6661 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 6721 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 6781 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 6841 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 6901 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 6961 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 7021 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 7081 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 7141 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 7201 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 7261 gaaatgttga atactcatat tcttcctt //