LOCUS Exported 7300 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-HLA-C-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7300) 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 7300) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7300 /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 4642..4686 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 4687..4704 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4844..4861) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4850..4961 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5079..5099) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5084..5413 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5264..5399 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5326..5345 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5461..5508 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5527..6126 /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(5527..5546) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6023..6043 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6256..6377 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6293..6312) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6347..6366 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6430..7290) /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 7053..7072 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7291..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 cttcctgaac 2821 tgctacgtgt ccggctttca ccccagcgac attgaggtgg acctgctgaa gaacggcgag 2881 cggatcgaga aggtggaaca cagcgatctg agcttcagca aggactggtc cttctacctg 2941 ctgtactaca ccgagttcac ccctaccgag aaggacgagt acgcctgcag agtgaaccac 3001 gtgacactga gccagcctaa gatcgtgaag tgggacagag acatgggcgg aggcggatct 3061 ggtggcggag gaagtggcgg cggaggatct tgtagccaca gcatgagata cttcgacacc 3121 gccgtgtcca gacctggaag aggcgagcct agattcatca gcgtgggcta cgtggacgac 3181 acccagttcg tcagattcga cagcgacgcc gcttctccca gaggggaacc tagagcacct 3241 tgggttgagc aagagggccc cgagtactgg gatagagaga cacagaagta caagcggcag 3301 gcccaggccg atagagtgtc cctgagaaac ctgcggggct actacaacca gagcgaggat 3361 ggctctcaca ccctgcagag aatgagcggc tgtgatctgg gccctgatgg cagactgctg 3421 agaggctatg atcagagcgc ctacgacggc aaggactata tcgccctgaa cgaggacctg 3481 cggtcttgga cagctgccga tacagctgcc cagattaccc agcggaaact ggaagccgcc 3541 agagccgctg aacagctgag agcttatctg gaaggcacct gtgtggaatg gctgcggaga 3601 tacctggaaa acggcaaaga gacactgcag cgggccgagc ctcctaagac acatgtgaca 3661 caccatcctc tgagcgacca cgaggccaca ctgagatgtt gggccctggg cttttaccct 3721 gccgagatca cactgacctg gcagagagat ggcgaggatc agacccagga taccgagctg 3781 gtggaaacaa gacctgccgg cgacggcacc ttccagaaat gggctgctgt ggtggtgcct 3841 agcggccaag agcagagata cacctgtcac atgcagcacg agggcctgca agagcctctg 3901 acactgtctt gggagcctag cagccagcca acaatcccca ttggcgcgcc gggcggcgac 3961 aagacccaca cctgcccccc ctgcccagct ccagaactgc tgggagggcc ttccgtgttc 4021 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc 4081 gtggtggtgg ccgtgtccca cgaggaccca gaggtgaagt tcaactggta cgtggacggc 4141 gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg 4201 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc 4261 aaagtctcca acaaggccct ggctgcccca atcgaaaaga caatcagcaa ggccaagggc 4321 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac 4381 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg 4441 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac 4501 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac 4561 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 4621 agcctgagcc ccggcggcag cggtcttaat gatattttcg aggctcagaa gattgagtgg 4681 catgaacacc accaccatca ccactaaggc cggcctgtag tcactggcac aacgcgttac 4741 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc 4801 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct 4861 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 4921 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct 4981 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 5041 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 5101 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 5161 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 5221 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 5281 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 5341 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 5401 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt 5461 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 5521 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg 5581 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat 5641 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg 5701 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg 5761 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg 5821 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc 5881 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt 5941 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc 6001 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc 6061 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt 6121 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 6181 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 6241 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6301 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 6361 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 6421 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 6481 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 6541 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc 6601 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 6661 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 6721 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat 6781 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6841 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6901 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6961 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7021 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7081 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7141 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7201 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7261 aagggcgaca cggaaatgtt gaatactcat attcttcctt //