LOCUS Exported 9106 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-SEMA4A-COMP5AP-AviTag-9xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9106) 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 9106) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9106 /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 4756..4779 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 6439..6483 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 6484..6510 /codon_start=1 /product="9xHis affinity tag" /label=9xHis /translation="HHHHHHHHH" primer_bind complement(6650..6667) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6656..6767 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6885..6905) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 6890..7219 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7070..7205 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7132..7151 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 7267..7314 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 7333..7932 /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(7333..7352) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7829..7849 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 8062..8183 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8099..8118) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8153..8172 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(8236..9096) /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 8859..8878 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9097..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 tggtggacaa 2761 ggacctatgc ctagagtgcg gtattacgcc ggcgacgaaa gaagggccct gagcttcttt 2821 caccagaagg gcctgcagga cttcgacaca ctgctgctga gcggagatgg caacacactg 2881 tatgtgggcg ccagagaagc cattctggcc ctggatattc aggaccctgg cgtgcccaga 2941 ctgaagaaca tgattccttg gcctgccagc gaccggaaga aaagcgagtg cgccttcaag 3001 aagaagtcca acgagacaca gtgcttcaac ttcatcagag tgctggtgtc ctacaacgtg 3061 acccacctgt acacctgtgg cacctttgcc ttttctcccg cctgcacctt catcgagctg 3121 caggattctt acctgctgcc tatcagcgag gacaaagtga tggaaggcaa gggccagtct 3181 cctttcgacc ctgctcacaa acataccgcc gtgctggtgg atggcatgct gtactccggc 3241 accatgaaca actttctggg cagcgagccc atcctgatga gaacactggg aagccagcct 3301 gtgctgaaaa ccgacaactt cctgagatgg ctgcaccacg atgccagctt tgtggccgcc 3361 attcctagca cacaggtggt gtactttttc ttcgaggaaa ccgccagcga gttcgacttc 3421 ttcgagagac tgcacaccag cagagtggcc agagtgtgca agaatgatgt cggcggagag 3481 aagctgctgc agaagaagtg gaccaccttc ctgaaggccc agctgctgtg tactcagcct 3541 ggccagctgc ctttcaacgt gatcagacat gctgtgctgc tgcccgccga ttctcctaca 3601 gctcctcaca tctacgccgt gttcacctct cagtggcaag tcggcggaac aagatcttcc 3661 gccgtgtgtg ccttcagcct gctggacatc gagagagtgt tcaagggcaa gtacaaagag 3721 ctgaacaaag aaaccagccg gtggacaacc tacagaggcc ccgagacaaa tcctagacct 3781 ggctcttgta gcgtgggccc tagctctgat aaggccctga cctttatgaa ggaccacttc 3841 ctcatggacg agcaggtcgt gggaacacca ctgctggtta agagcggcgt cgagtacaca 3901 agactggccg tggaaacagc ccagggcctc gatggacaca gccacctggt tatgtacctg 3961 ggcacaacaa ccggaagcct gcacaaagcc gtggtgtctg gcgattcttc tgcccacctg 4021 gtggaagaga tccagctgtt ccctgatcct gagcctgtgc ggaatctgca gcttgctcct 4081 acacagggcg ccgtgtttgt gggattttct ggcggagttt ggcgggtgcc aagagccaat 4141 tgcagcgtgt acgagagctg cgtggactgc gttctggcta gagatcctca ctgtgcttgg 4201 gaccctgaga gcagaacctg ttgtctgctg tctgccccta acctgaactc ctggaagcag 4261 gacatggaaa ggggcaatcc tgagtgggcc tgtgcctctg gacctatgag tagaagcctg 4321 aggccacaga gcagacccca gatcatcaaa gaggtgctgg ctgtccccaa cagcatcctg 4381 gaactgcctt gtcctcatct gagcgccctg gcctcttact attggagcca tggacctgct 4441 gctgtgccag aggccagctc tacagtgtac aatggcagcc tgctcctgat cgtgcaggat 4501 ggcgttggcg gactgtatca gtgttgggcc accgagaacg gcttcagcta ccctgtgatc 4561 agctactggg tcgacagcca ggatcagaca ctggctctcg atcctgaact ggccggcatt 4621 cccagagaac acgtgaaggt gcccctgaca agagttagtg gcggagctgc tctggctgcc 4681 cagcagtctt attggcctca tggcgcgccg gaagatcaag ttgatccaag attgattgat 4741 ggcaagggcg gatctctgga ggtgctgttc cagggaccag gcggaagcgg caatcccggc 4801 ggcgatctgg ccccccagat gctgcgcgag ctgcaggaga ccaatgccgc cctgcaggac 4861 gtgcgcgagc tgctgcgcca ccgcgtgaag gagatcacct tcctgaagaa caccgtgatg 4921 gagtgcgacg cctgcggtat gcagccctct agaggctccg gctccatcat cccagttgag 4981 gaggagaacc cggacttctg gaaccgcgag gcagccgagg ccctgggtgc cgccaagaag 5041 ctgcagcctg cacagacagc cgccaagaac ctcatcatct tcctgggcga tgggatgggg 5101 gtgtctacgg tgacagctgc caggatccta aaagggcaga agaaggacaa actggggcct 5161 gagatacccc tggccatgga ccgcttccca tatgtggctc tgtccaagac atacaatgta 5221 gacaaacatg tgccagacag tggagccaca gccacggcct acctgtgcgg ggtcaagggc 5281 aacttccaga ccattggctt gagtgcagcc gcccgcttta accagtgcaa cacgacacgc 5341 ggcaacgagg tcatctccgt gatgaatcgg gccaagaaag cagggaagtc agtgggagtg 5401 gtaaccacca cacgagtgca gcacgcctcg ccagccggca cctacgccca cacggtgaac 5461 cgcaactggt actcggacgc cgacgtgcct gcctcggccc gccaggaggg gtgccaggac 5521 atcgctacgc agctcatctc caacatggac attgacgtga tcctaggtgg aggccgaaag 5581 tacatgtttc gcatgggaac cccagaccct gagtacccag atgactacag ccaaggtggg 5641 accaggctgg acgggaagaa tctggtgcag gaatggctgg cgaagcgcca gggtgcccgg 5701 tatgtgtgga accgcactga gctcatgcag gcttccctgg acccgtctgt gacccatctc 5761 atgggtctct ttgagcctgg agacatgaaa tacgagatcc accgagactc cacactggac 5821 ccctccctga tggagatgac agaggctgcc ctgcgcctgc tgagcaggaa cccccgcggc 5881 ttcttcctct tcgtggaggg tggtcgcatc gaccatggtc atcatgaaag cagggcttac 5941 cgggcactga ctgagacgat catgttcgac gacgccattg agagggcggg ccagctcacc 6001 agcgaggagg acacgctgag cctcgtcact gccgaccact cccacgtctt ctccttcgga 6061 ggctaccccc tgcgagggag ctccatcttc gggctggccc ctggcaaggc ccgggacagg 6121 aaggcctaca cggtcctcct atacggaaac ggtccaggct atgtgctcaa ggacggcgcc 6181 cggccggatg ttaccgagag cgagagcggg agccccgagt atcggcagca gtcagcagtg 6241 cccctggacg aagagaccca cgcaggcgag gacgtggcgg tgttcgcgcg cggcccgcag 6301 gcgcacctgg ttcacggcgt gcaggagcag accttcatag cgcacgtcat ggccttcgcc 6361 gcctgcctgg agccctacac cgcctgcgac ctggcgcccc ccgccggcac caccgacgcc 6421 gcgcacccgg gtggcggagg actgaacgac atcttcgagg ctcagaaaat cgaatggcac 6481 gaacatcacc atcaccatca ccatcaccat taaggccggc ctgtagtcac tggcacaacg 6541 cgttacccgg taagccaatc gggtatacac ggtcgtcata ctgcagacag ggttcttcta 6601 ctttgcaaga tagtcttgag tagtaaaata aatagataga gaaaatcagc ctcgactgtg 6661 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 6721 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcaca acactcaacc 6781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 6841 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 6901 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 6961 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 7021 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 7081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 7141 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 7201 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 7261 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 7321 aggaactaaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 7381 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 7441 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 7501 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 7561 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 7621 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 7681 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 7741 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 7801 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 7861 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 7921 cccggtgcct gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg 7981 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 8041 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 8101 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 8161 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 8221 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 8281 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 8341 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga 8401 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 8461 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 8521 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt 8581 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 8641 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 8701 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 8761 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 8821 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 8881 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 8941 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 9001 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 9061 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctt //