LOCUS Exported 7708 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression of cell-surface protein extracellular domain fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells.. ACCESSION . VERSION . KEYWORDS pD649-HAsp-ULBP3-COMP5AP-AviTag-9xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7708) 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 7708) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7708 /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 3358..3381 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 5041..5085 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 5086..5112 /codon_start=1 /product="9xHis affinity tag" /label=9xHis /translation="HHHHHHHHH" primer_bind complement(5252..5269) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5258..5369 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5487..5507) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5492..5821 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5672..5807 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5734..5753 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5869..5916 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5935..6534 /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(5935..5954) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6431..6451 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6664..6785 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6701..6720) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6755..6774 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6838..7698) /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 7461..7480 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7699..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 cggccgccga tgctcacagc 2761 ctgtggtaca acttcaccat catccatctg cctcggcacg gacagcagtg gtgcgaagtg 2821 cagtctcagg tggaccagaa gaacttcctg agctacgact gcggcagcga caaggtgctg 2881 tctatgggac acctggaaga acagctgtac gccacagatg cctggggcaa gcagctggaa 2941 atgctgagag aagtgggcca gagactgaga ctggaactgg ccgataccga gctggaagat 3001 ttcacaccta gcggccctct gacactgcaa gtgcggatga gctgcgaatg cgaggccgat 3061 ggctatatca gaggcagctg gcagttcagc ttcgacggca gaaagttcct gctgttcgac 3121 agcaacaacc ggaagtggac agtggtgcat gccggcgcta gacgcatgaa ggaaaagtgg 3181 gagaaagact ccggcctgac caccttcttc aagatggtgt ccatgcggga ctgcaagagc 3241 tggctgagag acttcctgat gcaccggaag aagcggctgg aacctaccgc tcctcctacc 3301 atgggcgcgc cggaagatca agttgatcca agattgattg atggcaaggg cggatctctg 3361 gaggtgctgt tccagggacc aggcggaagc ggcaatcccg gcggcgatct ggccccccag 3421 atgctgcgcg agctgcagga gaccaatgcc gccctgcagg acgtgcgcga gctgctgcgc 3481 caccgcgtga aggagatcac cttcctgaag aacaccgtga tggagtgcga cgcctgcggt 3541 atgcagccct ctagaggctc cggctccatc atcccagttg aggaggagaa cccggacttc 3601 tggaaccgcg aggcagccga ggccctgggt gccgccaaga agctgcagcc tgcacagaca 3661 gccgccaaga acctcatcat cttcctgggc gatgggatgg gggtgtctac ggtgacagct 3721 gccaggatcc taaaagggca gaagaaggac aaactggggc ctgagatacc cctggccatg 3781 gaccgcttcc catatgtggc tctgtccaag acatacaatg tagacaaaca tgtgccagac 3841 agtggagcca cagccacggc ctacctgtgc ggggtcaagg gcaacttcca gaccattggc 3901 ttgagtgcag ccgcccgctt taaccagtgc aacacgacac gcggcaacga ggtcatctcc 3961 gtgatgaatc gggccaagaa agcagggaag tcagtgggag tggtaaccac cacacgagtg 4021 cagcacgcct cgccagccgg cacctacgcc cacacggtga accgcaactg gtactcggac 4081 gccgacgtgc ctgcctcggc ccgccaggag gggtgccagg acatcgctac gcagctcatc 4141 tccaacatgg acattgacgt gatcctaggt ggaggccgaa agtacatgtt tcgcatggga 4201 accccagacc ctgagtaccc agatgactac agccaaggtg ggaccaggct ggacgggaag 4261 aatctggtgc aggaatggct ggcgaagcgc cagggtgccc ggtatgtgtg gaaccgcact 4321 gagctcatgc aggcttccct ggacccgtct gtgacccatc tcatgggtct ctttgagcct 4381 ggagacatga aatacgagat ccaccgagac tccacactgg acccctccct gatggagatg 4441 acagaggctg ccctgcgcct gctgagcagg aacccccgcg gcttcttcct cttcgtggag 4501 ggtggtcgca tcgaccatgg tcatcatgaa agcagggctt accgggcact gactgagacg 4561 atcatgttcg acgacgccat tgagagggcg ggccagctca ccagcgagga ggacacgctg 4621 agcctcgtca ctgccgacca ctcccacgtc ttctccttcg gaggctaccc cctgcgaggg 4681 agctccatct tcgggctggc ccctggcaag gcccgggaca ggaaggccta cacggtcctc 4741 ctatacggaa acggtccagg ctatgtgctc aaggacggcg cccggccgga tgttaccgag 4801 agcgagagcg ggagccccga gtatcggcag cagtcagcag tgcccctgga cgaagagacc 4861 cacgcaggcg aggacgtggc ggtgttcgcg cgcggcccgc aggcgcacct ggttcacggc 4921 gtgcaggagc agaccttcat agcgcacgtc atggccttcg ccgcctgcct ggagccctac 4981 accgcctgcg acctggcgcc ccccgccggc accaccgacg ccgcgcaccc gggtggcgga 5041 ggactgaacg acatcttcga ggctcagaaa atcgaatggc acgaacatca ccatcaccat 5101 caccatcacc attaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa 5161 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg 5221 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca 5281 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 5341 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt 5401 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 5461 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 5521 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 5581 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 5641 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 5701 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 5761 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 5821 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa 5881 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc 5941 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc 6001 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac 6061 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc 6121 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc 6181 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg 6241 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg 6301 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc 6361 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca 6421 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc 6481 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg 6541 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 6601 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 6661 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 6721 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 6781 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta 6841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 6901 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 6961 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca 7021 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 7081 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 7141 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 7201 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 7261 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 7321 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 7381 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 7441 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 7501 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 7561 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 7621 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 7681 gaaatgttga atactcatat tcttcctt //