LOCUS Exported 7605 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION NanoLuc Gateway shuttle vector for N-terminal fusions. ACCESSION . VERSION . KEYWORDS pcDNA3.1 myc-NL-GW SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7605) AUTHORS Trepte P, Kruse S, Kostova S, Hoffmann S, Buntru A, Tempelmeier A, Secker C, Diez L, Schulz A, Klockmeier K, Zenkner M, Golusik S, Rau K, Schnoegl S, Garner CC, Wanker EE. TITLE LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells JOURNAL Molecular Systems Biology (2018) 14, e8071 REFERENCE 2 (bases 1 to 7605) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7605 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 907..936 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 937..1446 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILA" protein_bind 1453..1577 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 1602..1632 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 1686..2345 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(1752..1771) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 2687..2992 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2871..2890 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(3033..3157) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3199..3216) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3205..3429 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3475..3903 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3562..3581) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3772..3793 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3912..3932) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3917..4246 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4097..4232 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4159..4178 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4313..5107 /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4367..4386) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4977..4996 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5281..5402 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5318..5337) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5372..5391 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5451..5467) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5451..5467) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5464..5486) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5475..5491 /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(5499..5529) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5544..5565 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5682..5699) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5853..6438) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5933..5952) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6609..7469) /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 7232..7251 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7470..7574) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 accatggaac agaaactgat ctctgaagaa gacctggtct tcacactcga agatttcgtt 961 ggggactggc gacagacagc cggctacaac ctggaccaag tccttgaaca gggaggtgtg 1021 tccagtttgt ttcagaatct cggggtgtcc gtaactccga tccaaaggat tgtcctgagc 1081 ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc cgtatgaagg tctgagcggc 1141 gaccaaatgg gccagatcga aaaaattttt aaggtggtgt accctgtgga tgatcatcac 1201 tttaaggtga tcctgcacta tggcacactg gtaatcgacg gggttacgcc gaacatgatc 1261 gactatttcg gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta 1321 acagggaccc tgtggaacgg caacaaaatt atcgacgagc gcctgatcaa ccccgacggc 1381 tccctgctgt tccgagtaac catcaacgga gtgaccggct ggcggctgtg cgaacgcatt 1441 ctggcgaagc ttacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat 1501 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa 1561 catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg 1621 gctcgtataa tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag 1681 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 1741 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 1801 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 1861 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag 1921 acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa 1981 ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 2041 tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 2101 ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 2161 acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 2221 aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc tgtgatggct 2281 tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 2341 cgtaatctag aggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg 2401 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggtg 2461 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc 2521 atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt 2581 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt 2641 attgaaatga acggctcttt tgctgacgag aacagggact ggtgaaatgc agtttaaggt 2701 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat 2761 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa 2821 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac 2881 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca 2941 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg 3001 ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac 3061 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat 3121 tttacgtttc tcgttcagct ttcttgtaca aagtggtgta actcgagtct agagggcccg 3181 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 3241 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 3301 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 3361 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 3421 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 3481 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 3541 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 3601 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 3661 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 3721 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 3781 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 3841 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 3901 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 3961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 4021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 4081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 4141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt 4201 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc 4261 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga 4321 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 4381 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 4441 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga 4501 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 4561 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 4621 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 4681 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 4741 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 4801 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga 4861 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 4921 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 4981 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 5041 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 5101 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca 5161 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 5221 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 5281 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 5341 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 5401 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 5461 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 5521 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 5581 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5641 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5701 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5761 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5821 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5881 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5941 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 6001 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 6061 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 6121 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 6181 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 6241 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 6301 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 6361 tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc 6421 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 6481 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 6541 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 6601 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 6661 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 6721 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 6781 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 6841 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6901 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 6961 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 7021 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 7081 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 7141 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 7201 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 7261 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 7321 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 7381 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 7441 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 7501 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 7561 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc //