LOCUS Exported 8035 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-SEMA4C-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8035) 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 8035) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8035 /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 5377..5421 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 5422..5439 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5579..5596) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5585..5696 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5814..5834) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5819..6148 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5999..6134 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6061..6080 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 6196..6243 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 6262..6861 /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(6262..6281) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6758..6778 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6991..7112 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7028..7047) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7082..7101 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(7165..8025) /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 7788..7807 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8026..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 cggccgccgc tgaagtttgg 2761 tggaatctgg tgcccagaaa gaccgtgtct agcggcgaac tggccacagt cgtgcggaga 2821 ttttcccaga ccggcatcca ggactttctg accctgacac tgaccgagcc taccggcctg 2881 ctgtatgtgg gagctagaga agccctgttc gccttcagca tggaagccct ggaactgcag 2941 ggcgccatct cttgggaagc ccctgtggaa aagaaaaccg agtgcatcca gaagggcaag 3001 aacaaccaga ccgagtgctt caacttcatc cggtttctgc agccctacaa cgccagccac 3061 ctgtacgtgt gtggcaccta cgccttccag cctaagtgca cctacgtgaa catgctgacc 3121 ttcacactgg aacacggcga gttcgaggac ggcaagggca agtgtcctta cgatcctgcc 3181 aaaggccatg ccggactgct ggttgatggc gagctgtact ctgctaccct gaacaacttc 3241 ctgggcaccg agccaatcat cctgcggaat atgggccctc accacagcat gaagaccgag 3301 tacctggcct tctggctgaa cgagcctcac tttgtgggca gcgcctatgt gcctgagagc 3361 gtgggatctt tcaccggcga cgacgacaag gtgtacttct tcttccgcga acgcgccgtg 3421 gaaagcgact gttatgctga acaggtggtg gccagagtgg ctagagtgtg caagggcgat 3481 atgggcggag ccagaacact gcagagaaag tggaccacct tcctgaaggc cagactggct 3541 tgcagcgccc ctaattggca gctgtatttc aaccagctgc aggccatgca caccctgcag 3601 gataccagct ggcacaacac cacctttttc ggagtgtttc aggcccagtg gggcgacatg 3661 tacctgtctg ccatctgcga gtatcagctg gaagagatcc agcgggtgtt cgagggcccc 3721 tacaaagagt atcacgaaga ggcccagaag tgggacagat acacagaccc cgtgccttct 3781 ccaagacctg gcagctgcat caacaactgg cacagacggc acggctacac ctctagcctg 3841 gaactccccg acaacatcct gaacttcgtg aagaaacacc ctctgatgga agaacaagtc 3901 ggccccagat ggtccagacc tctgctggtt aagaagggca ccaacttcac ccacctggtg 3961 gccgatagag tgacaggact ggatggcgcc acctacaccg tgctgtttat cggaacaggc 4021 gacggctggc tgctcaaggc tgtttctctt ggaccttggg tgcacctgat cgaagaactg 4081 caactgttcg accaagagcc tatgcggagc ctggtgctga gccagtctaa gaagctgctg 4141 tttgccggca gcagatccca gctggttcag ctgcctgtgg ccgactgcat gaagtacaga 4201 tcctgcgccg attgcgtgct ggccagagat ccttactgtg cttggagcgt gaacaccagc 4261 agatgtgtgg ccgttggcgg acacagtgga agcctgctga ttcagcacgt gatgaccagc 4321 gacaccagcg gcatctgcaa tctgcggggc tctaagaaag tgcggcccac acctaagaac 4381 atcacagtgg tggccggcac cgatctggtg ctgccttgtc acctgagcag caatctggcc 4441 cacgccagat ggacattcgg cggcagagat ctgccagctg agcagcctgg ctccttcctg 4501 tatgatgctc gactgcaggc cctggtggtt atggctgcac agcctagaca tgccggcgct 4561 taccactgct tcagcgagga acaaggcgct agactggccg ccgaaggata tctggtggct 4621 gtggttgctg gccctagcgt gacactggaa gctagagcac ccctggaaaa cctcggaggc 4681 gcgccgggcg gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga 4741 gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 4801 cccgaggtga cctgcgtggt ggtggccgtg tcccacgagg acccagaggt gaagttcaac 4861 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac 4921 aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 4981 aaagaataca agtgcaaagt ctccaacaag gccctggctg ccccaatcga aaagacaatc 5041 agcaaggcca agggccagcc acgggagccc caggtgtaca ccctgccccc cagccgggag 5101 gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat 5161 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca 5221 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagtccagg 5281 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 5341 acccagaagt ccctgagcct gagccccggc ggcagcggtc ttaatgatat tttcgaggct 5401 cagaagattg agtggcatga acaccaccac catcaccact aaggccggcc tgtagtcact 5461 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg 5521 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc 5581 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 5641 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa 5701 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 5761 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 5821 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 5881 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 5941 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 6001 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 6061 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 6121 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 6181 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac 6241 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc 6301 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 6361 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 6421 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 6481 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 6541 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg 6601 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 6661 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc 6721 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg 6781 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 6841 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat 6901 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 6961 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 7021 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 7081 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 7141 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc 7201 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7261 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc 7321 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7381 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7441 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg 7501 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7561 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7621 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7681 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7741 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7801 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7861 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7921 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7981 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt //