LOCUS Exported 8059 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-SEMA4A-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8059) 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 8059) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8059 /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 5401..5445 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 5446..5463 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5603..5620) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5609..5720 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5838..5858) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5843..6172 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6023..6158 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6085..6104 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 6220..6267 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 6286..6885 /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(6286..6305) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6782..6802 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 7015..7136 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7052..7071) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7106..7125 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(7189..8049) /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 7812..7831 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8050..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 ggcggcgaca agacccacac ctgccccccc 4741 tgcccagctc cagaactgct gggagggcct tccgtgttcc tgttcccccc caagcccaag 4801 gacaccctga tgatcagcag gacccccgag gtgacctgcg tggtggtggc cgtgtcccac 4861 gaggacccag aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag 4921 accaagccca gagaggagca gtacaacagc acctacaggg tggtgtccgt gctgaccgtg 4981 ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aagtctccaa caaggccctg 5041 gctgccccaa tcgaaaagac aatcagcaag gccaagggcc agccacggga gccccaggtg 5101 tacaccctgc cccccagccg ggaggagatg accaagaacc aggtgtccct gacctgtctg 5161 gtgaagggct tctaccccag cgatatcgcc gtggagtggg agagcaacgg ccagcccgag 5221 aacaactaca agaccacccc cccagtgctg gacagcgacg gcagcttctt cctgtacagc 5281 aagctgaccg tggacaagtc caggtggcag cagggcaacg tgttcagctg cagcgtgatg 5341 cacgaggccc tgcacaacca ctacacccag aagtccctga gcctgagccc cggcggcagc 5401 ggtcttaatg atattttcga ggctcagaag attgagtggc atgaacacca ccaccatcac 5461 cactaaggcc ggcctgtagt cactggcaca acgcgttacc cggtaagcca atcgggtata 5521 cacggtcgtc atactgcaga cagggttctt ctactttgca agatagtctt gagtagtaaa 5581 ataaatagat agagaaaatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 5641 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 5701 taaaatgagg aaattgcatc acaacactca accctatctc ggtctattct tttgatttat 5761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5821 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 5881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 5941 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 6001 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 6061 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga 6121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc 6181 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca 6241 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatgac cgagtacaag 6301 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 6361 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 6421 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 6481 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 6541 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 6601 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 6661 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 6721 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac atccgcgccc 6781 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 6841 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacacgt gctacgagat 6901 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 6961 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 7021 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 7081 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 7141 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt 7201 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 7261 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat 7321 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg 7381 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 7441 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 7501 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 7561 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 7621 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 7681 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 7741 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 7801 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 7861 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 7921 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 7981 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 8041 aatactcata ttcttcctt //