LOCUS Exported 6721 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-TNFSF9-Fc(DAPA)-AviTag-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6721) 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 6721) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6721 /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 4063..4107 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 4108..4125 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4265..4282) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4271..4382 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4500..4520) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4505..4834 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4685..4820 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4747..4766 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 4882..4929 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 4948..5547 /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(4948..4967) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5444..5464 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5677..5798 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5714..5733) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5768..5787 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(5851..6711) /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 6474..6493 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6712..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 ttgtccttgg 2761 gctgtttctg gtgctagagc ctctcctgga tctgccgcct ctcctagact gagagaggga 2821 cctgagctga gccctgatga tcctgctgga ctgctggatc tgagacaggg catgtttgct 2881 cagctggtgg cccagaacgt gctgctgatt gatggccctc tgagctggta ctctgatcct 2941 ggacttgctg gcgttagcct gaccggcgga ctgagctaca aagaggacac caaagaactg 3001 gtggtggcca aggccggcgt gtactacgtg ttctttcagc tggaactgcg gagagtggtg 3061 gccggcgaag gatctggatc tgtgtctctg gctctgcatc tgcagcctct gagatctgct 3121 gctggtgctg ctgctctggc actgacagtt gatctgcctc ctgccagcag cgaggccaga 3181 aactctgcct ttggcttcca aggcagactg ctgcacctgt ctgctggaca gagactggga 3241 gtgcacctcc acacagaagc cagagctaga cacgcttggc agctgacaca aggcgctaca 3301 gtgctgggcc tgtttagagt gacacctgag attccagccg gcctgccttc tccaagaagc 3361 gaaggcgcgc cgggcggcga caagacccac acctgccccc cctgcccagc tccagaactg 3421 ctgggagggc cttccgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 3481 aggacccccg aggtgacctg cgtggtggtg gccgtgtccc acgaggaccc agaggtgaag 3541 ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagagaggag 3601 cagtacaaca gcacctacag ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg 3661 aacggcaaag aatacaagtg caaagtctcc aacaaggccc tggctgcccc aatcgaaaag 3721 acaatcagca aggccaaggg ccagccacgg gagccccagg tgtacaccct gccccccagc 3781 cgggaggaga tgaccaagaa ccaggtgtcc ctgacctgtc tggtgaaggg cttctacccc 3841 agcgatatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc 3901 cccccagtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 3961 tccaggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 4021 cactacaccc agaagtccct gagcctgagc cccggcggca gcggtcttaa tgatattttc 4081 gaggctcaga agattgagtg gcatgaacac caccaccatc accactaagg ccggcctgta 4141 gtcactggca caacgcgtta cccggtaagc caatcgggta tacacggtcg tcatactgca 4201 gacagggttc ttctactttg caagatagtc ttgagtagta aaataaatag atagagaaaa 4261 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 4321 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 4381 tcacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 4441 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg 4501 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 4561 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 4621 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 4681 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 4741 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg 4801 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat 4861 tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta 4921 taatacgaca aggtgaggaa ctaaaccatg accgagtaca agcccacggt gcgcctcgcc 4981 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 5041 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 5101 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 5161 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 5221 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 5281 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 5341 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 5401 gagcgcgccg gggtgcccgc cttcctggag acatccgcgc cccgcaacct ccccttctac 5461 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 5521 tgcatgaccc gcaagcccgg tgcctgacac gtgctacgag atttcgattc caccgccgcc 5581 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 5641 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 5701 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 5761 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 5821 tctagctaga gcttggcgta atcatggtca ttaccaatgc ttaatcagtg aggcacctat 5881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5941 tacgatacgg gagggcttac catctggccc cagcgctgcg atgataccgc gagaaccacg 6001 ctcaccggct ccggatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt 6181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tattcttcct 6721 t //