LOCUS Exported 7684 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-TNF-COMP5AP-AviTag-9xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7684) 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 7684) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7684 /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 3334..3357 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 5017..5061 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 5062..5088 /codon_start=1 /product="9xHis affinity tag" /label=9xHis /translation="HHHHHHHHH" primer_bind complement(5228..5245) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5234..5345 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5463..5483) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5468..5797 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5648..5783 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5710..5729 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5845..5892 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5911..6510 /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(5911..5930) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6407..6427 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6640..6761 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6677..6696) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6731..6750 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6814..7674) /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 7437..7456 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7675..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 cggccgccgg acctcagaga 2761 gaggaatttc ctagagatct gagcctgatc agccctctgg ctcaggccgt cagaagcagc 2821 agcagaacac ctagcgataa gcccgtggct cacgtggtgg ctaatcctca ggctgaagga 2881 cagctgcagt ggctgaatag aagggccaac gctctgctgg ccaacggcgt ggaactgaga 2941 gataatcagc tggtggtgcc cagcgagggc ctgtacctga tctatagcca ggtgctgttc 3001 aaaggccagg gctgcccttc tacacacgtg ctgctgaccc acaccatcag cagaatcgcc 3061 gtgtcctacc agaccaaagt gaacctgctg agcgccatca agagcccctg tcagagagaa 3121 acacctgagg gcgccgaagc caagccttgg tacgaaccta tctatctcgg cggcgtgttc 3181 cagctcgaga agggcgatag actgagcgcc gagatcaaca gacccgacta cctggatttt 3241 gccgagagcg gccaggtgta cttcggcatc attgcccttg gcgcgccgga agatcaagtt 3301 gatccaagat tgattgatgg caagggcgga tctctggagg tgctgttcca gggaccaggc 3361 ggaagcggca atcccggcgg cgatctggcc ccccagatgc tgcgcgagct gcaggagacc 3421 aatgccgccc tgcaggacgt gcgcgagctg ctgcgccacc gcgtgaagga gatcaccttc 3481 ctgaagaaca ccgtgatgga gtgcgacgcc tgcggtatgc agccctctag aggctccggc 3541 tccatcatcc cagttgagga ggagaacccg gacttctgga accgcgaggc agccgaggcc 3601 ctgggtgccg ccaagaagct gcagcctgca cagacagccg ccaagaacct catcatcttc 3661 ctgggcgatg ggatgggggt gtctacggtg acagctgcca ggatcctaaa agggcagaag 3721 aaggacaaac tggggcctga gatacccctg gccatggacc gcttcccata tgtggctctg 3781 tccaagacat acaatgtaga caaacatgtg ccagacagtg gagccacagc cacggcctac 3841 ctgtgcgggg tcaagggcaa cttccagacc attggcttga gtgcagccgc ccgctttaac 3901 cagtgcaaca cgacacgcgg caacgaggtc atctccgtga tgaatcgggc caagaaagca 3961 gggaagtcag tgggagtggt aaccaccaca cgagtgcagc acgcctcgcc agccggcacc 4021 tacgcccaca cggtgaaccg caactggtac tcggacgccg acgtgcctgc ctcggcccgc 4081 caggaggggt gccaggacat cgctacgcag ctcatctcca acatggacat tgacgtgatc 4141 ctaggtggag gccgaaagta catgtttcgc atgggaaccc cagaccctga gtacccagat 4201 gactacagcc aaggtgggac caggctggac gggaagaatc tggtgcagga atggctggcg 4261 aagcgccagg gtgcccggta tgtgtggaac cgcactgagc tcatgcaggc ttccctggac 4321 ccgtctgtga cccatctcat gggtctcttt gagcctggag acatgaaata cgagatccac 4381 cgagactcca cactggaccc ctccctgatg gagatgacag aggctgccct gcgcctgctg 4441 agcaggaacc cccgcggctt cttcctcttc gtggagggtg gtcgcatcga ccatggtcat 4501 catgaaagca gggcttaccg ggcactgact gagacgatca tgttcgacga cgccattgag 4561 agggcgggcc agctcaccag cgaggaggac acgctgagcc tcgtcactgc cgaccactcc 4621 cacgtcttct ccttcggagg ctaccccctg cgagggagct ccatcttcgg gctggcccct 4681 ggcaaggccc gggacaggaa ggcctacacg gtcctcctat acggaaacgg tccaggctat 4741 gtgctcaagg acggcgcccg gccggatgtt accgagagcg agagcgggag ccccgagtat 4801 cggcagcagt cagcagtgcc cctggacgaa gagacccacg caggcgagga cgtggcggtg 4861 ttcgcgcgcg gcccgcaggc gcacctggtt cacggcgtgc aggagcagac cttcatagcg 4921 cacgtcatgg ccttcgccgc ctgcctggag ccctacaccg cctgcgacct ggcgcccccc 4981 gccggcacca ccgacgccgc gcacccgggt ggcggaggac tgaacgacat cttcgaggct 5041 cagaaaatcg aatggcacga acatcaccat caccatcacc atcaccatta aggccggcct 5101 gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg tcgtcatact 5161 gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa tagatagaga 5221 aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 5281 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 5341 gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 5401 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct 5461 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 5521 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 5581 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 5641 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 5701 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 5761 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 5821 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata 5881 gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac ggtgcgcctc 5941 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 6001 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 6061 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 6121 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 6181 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 6241 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 6301 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 6361 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc 6421 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 6481 tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga ttccaccgcc 6541 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 6601 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 6661 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 6721 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 6781 acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca gtgaggcacc 6841 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6901 aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc 6961 acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 7021 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 7081 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt 7141 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 7201 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 7261 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 7321 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 7381 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 7441 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 7501 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 7561 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 7621 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt 7681 cctt //