LOCUS Exported 8201 bp ds-DNA circular SYN 09-DEC-2022 DEFINITION Expression of SNAPf and mXFPm-tagged IFNAR1 at the plasma membrane, e.g. for single molecule fluorescence microscopy. ACCESSION . VERSION . KEYWORDS pSems-leader-SNAPf-mXFPm-IFNAR1(28-557) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8201) AUTHORS Sotolongo Bellon J, Birkholz O, Richter CP, Eull F, Kenneweg H, Wilmes S, Rothbauer U, You C, Walter MR, Kurre R, Piehler J TITLE Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane. JOURNAL Cell Rep Methods. 2022 Feb 4;2(2):100165. doi: 10.1016/j.crmeth.2022.100165. eCollection 2022 Feb 28. PUBMED 35474965 REFERENCE 2 (bases 1 to 8201) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 9, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8201 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 137..516 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 517..720 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 671..691 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 765..784 /label=T7 /note="T7 promoter, forward primer" promoter 765..783 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" sig_peptide 820..882 /product="leader sequence from mouse immunoglobulin kappa light chain" /label=Ig-kappa leader CDS 883..909 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 919..1464 /codon_start=1 /product="human O6-alkylguanine-DNA-alkyltransferase variant that forms covalent bonds with benzylguanine derivatives" /label=SNAP-tag(R) /note="fast-reacting variant, optimized for mammalian expression" /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA VKEWLLAHEGHRLGKPGLG" CDS 1486..2202 /codon_start=1 /product="enhanced CFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTFGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLKYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1531..1552) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1792..1811) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2139..2160 /label=EGFP-C /note="EGFP, forward primer" intron 3879..4108 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 4091..4110 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 4181..4754 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4348..4365) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4575..4594 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4790..5593 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4853..4872) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5463..5482 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5723..5844 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5760..5779) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(5946..5962) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5946..5962) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5959..5981) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5970..5986 /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)." promoter complement(5994..6024) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6039..6060 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6177..6194) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6348..6936) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6428..6447) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7107..7967) /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 7730..7749 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7968..8072) /gene="bla" /label=AmpR promoter primer_bind complement(8147..8166) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc 61 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg 121 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 721 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat 781 agggagaccc aagcttggta ccgagctcgg atcgatatca tggagacaga cacactcctg 841 ctatgggtac tgctgctctg ggttccaggt tccactggtg actatccata tgatgttcca 901 gattatgctg aattcaccat ggacaaagac tgcgaaatga agcgcaccac cctggatagc 961 cctctgggca agctggaact gtctgggtgc gaacagggcc tgcaccgtat catcttcctg 1021 ggcaaaggaa catctgccgc cgacgccgtg gaagtgcctg ccccagccgc cgtgctgggc 1081 ggaccagagc cactgatgca ggccaccgcc tggctcaacg cctactttca ccagcctgag 1141 gccatcgagg agttccctgt gccagccctg caccacccag tgttccagca ggagagcttt 1201 acccgccagg tgctgtggaa actgctgaaa gtggtgaagt tcggagaggt catcagctac 1261 agccacctgg ccgccctggc cggcaatccc gccgccaccg ccgccgtgaa aaccgccctg 1321 agcggaaatc ccgtgcccat tctgatcccc tgccaccggg tggtgcaggg cgacctggac 1381 gtggggggct acgagggcgg gctcgccgtg aaagagtggc tgctggccca cgagggccac 1441 agactgggca agcctgggct gggtcctgca ggtataggcg cgcccatggt gagcaagggc 1501 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 1561 cacaggttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 1621 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 1681 acctttggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 1741 aagtccgcca tgcccgaagg ctacgtccag gagcgtacca tcttcttcaa ggacgacggc 1801 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 1861 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct gaaatacaac 1921 tacatcagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac 1981 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 2041 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 2101 tccaaactga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 2161 accgccgccg ggatcactct cggcatggac gagctgtaca agggatccct cgagtctaaa 2221 aatctaaaat ctcctcaaaa agtagaggtc gacatcatag atgacaactt tatcctgagg 2281 tggaacagga gcgatgagtc tgtcgggaat gtgacttttt cattcgatta tcaaaaaact 2341 gggatggata attggataaa attgtctggg tgtcagaata ttactagtac caaatgcaac 2401 ttttcttcac tcaagctgaa tgtttatgaa gaaattaaat tgcgtataag agcagaaaaa 2461 gaaaacactt cttcatggta tgaggttgac tcatttacac catttcgcaa agctcagatt 2521 ggtcctccag aagtacattt agaagctgaa gataaggcaa tagtgataca catctctcct 2581 ggaacaaaag atagtgttat gtgggctttg gatggtttaa gctttacata tagcttactt 2641 atctggaaaa actcttcagg tgtagaagaa aggattgaaa atatttattc cagacataaa 2701 atttataaac tctcaccaga gactacttat tgtctaaaag ttaaagcagc actacttacg 2761 tcatggaaaa ttggtgtcta tagtccagta cattgtataa agaccacagt tgaaaatgaa 2821 ctacctccac cagaaaatat agaagtcagt gtccaaaatc agaactatgt tcttaaatgg 2881 gattatacat atgcaaacat gacctttcaa gttcagtggc tccacgcctt tttaaaaagg 2941 aatcctggaa accatttgta taaatggaaa caaatacctg actgtgaaaa tgtcaaaact 3001 acccagtgtg tctttcctca aaacgttttc caaaaaggaa tttaccttct ccgcgtacaa 3061 gcatctgatg gaaataacac atctttttgg tctgaagaga taaagtttga tactgaaata 3121 caagctttcc tacttcctcc agtctttaac attagatccc ttagtgattc attccatatc 3181 tatatcggtg ctccaaaaca gtctggaaac acgcctgtga tccaggatta tccactgatt 3241 tatgaaatta ttttttggga aaacacttca aatgctgaga gaaaaattat cgagaaaaaa 3301 actgatgtta cagttcctaa tttgaaacca ctgactgtat attgtgtgaa agccagagca 3361 cacaccatgg atgaaaagct gaataaaagc agtgttttta gtgacgctgt atgtgagaaa 3421 acaaaaccag gaaatacctc taaaatttgg cttatagttg gaatttgtat tgcattattt 3481 gctctcccgt ttgtcattta tgctgcgaaa gtcttcttga gatgcatcaa ttatgtcttc 3541 tttccatcac ttaaaccttc ttccagtata gatgagtatt tctctgaaca gccattgaag 3601 aatcttctgc tttcaacttc tgaggaacaa atcgaaaaat gtttcataat tgaaaatata 3661 agcacaattg ctacagtaga agaaactaat caaactgatg aagatcataa aaaatacagt 3721 tcccaaacta gccaagattc aggaaattat tctaatgaag atgaaagcga aagtaaaaca 3781 agtgaagaac tacagcagga ctttgtatga gcggccgcat agataactga tccagtgtgc 3841 tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc tctcaaaagc 3901 gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac 3961 ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa agacaatctt 4021 tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt gagtgacaat 4081 gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact gcaggtcgag 4141 catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctctccc tccccccccc 4201 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat 4261 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct 4321 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg 4381 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc 4441 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg 4501 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg 4561 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga 4621 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt 4681 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa 4741 aaacacgatg ataagcttgc cacaacccgg gataattcct gcagccaata tgggatcggc 4801 cattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4861 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4921 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4981 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 5041 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 5101 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 5161 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 5221 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 5281 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 5341 cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 5401 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 5461 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 5521 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 5581 cgagttcttc tgaggggatc aattctctag ataactgatc ataatcagcc ataccacatt 5641 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 5701 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 5761 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 5821 gtccaaactc atcaatgtat cttaacgcgt cgagtgcatt ctagttgtgg tttgtccaaa 5881 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 5941 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 6001 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 6061 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 6121 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 6181 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6241 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6301 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6361 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6421 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6481 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6541 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6601 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6661 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6721 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6781 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6841 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6901 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6961 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 7021 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 7081 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 7141 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 7201 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 7261 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 7321 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 7381 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7441 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7501 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7561 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7621 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7681 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7741 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7801 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7861 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7921 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7981 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 8041 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 8101 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 8161 atgccgcata gttaagccag tatctgctcc ctgcttgtgt g //