LOCUS Exported 7962 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gateway-compatible mammalian expression destination vector for creating SFB N-terminal fusions (S protein tag, Flag tag, Streptavidin binding peptide). ACCESSION . VERSION . KEYWORDS pMH-SFB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7962) AUTHORS An L, Jiang Y, Ng HH, Man EP, Chen J, Khoo US, Gong Q, Huen MS TITLE Dual-utility NLS drives RNF169-dependent DNA damage responses. JOURNAL Proc Natl Acad Sci U S A. 2017 Apr 4;114(14):E2872-E2881. doi: 10.1073/pnas.1616602114. Epub 2017 Mar 21. PUBMED 28325877 REFERENCE 2 (bases 1 to 7962) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7962 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 22..325 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 326..529 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 480..500 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 589..633 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 667..690 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 694..717 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 727..840 /codon_start=1 /product="streptavidin-binding peptide" /label=SBP /note="selected from a peptide library; binds streptavidin with nanomolar affinity (Keefe et al., 2001)" /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP" protein_bind 913..1037 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 1062..1092 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 1146..1826 /codon_start=1 /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE DPAY" primer_bind complement(1212..1231) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 2146..2451 /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 VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2330..2349 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(2492..2616) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(2799..2816) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind complement(2878..2898) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2883..3212 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3063..3198 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3125..3144 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3222..3821 /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(3222..3241) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3718..3738 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 4042..4511 /note="long terminal repeat from Moloney murine leukemia virus" primer_bind complement(4641..4658) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4812..5400) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4892..4911) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5560..6420) /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 6183..6202 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6421..6525) /gene="bla" /label=AmpR promoter LTR 6556..7025 /note="long terminal repeat from Moloney murine leukemia virus" misc_feature 7090..7289 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 7490..7906 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 7836..7858 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 7874..7890 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" ORIGIN 1 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 61 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 121 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 181 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 241 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 301 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 361 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 421 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 481 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa 541 ccgtcagatc cgctagcgct accggactca gatctcgagg ccaccatgaa agaaaccgct 601 gctgctaaat tcgaacgcca gcacatggac agcggagccg gcgcaggagc cggcgctgac 661 gcgcctgact acaaagacga tgacgacaag ggagattaca aggatgacga tgacaaaggc 721 gcgtcgatgg acgagaagac caccggctgg cggggcggcc acgtggtgga gggcctggcc 781 ggcgagctgg agcagctgcg ggccaggctg gagcaccacc ctcagggcca gcgggagccc 841 tccggcggct gcaagctggg cggcgcgcct gaattctgca gtcgacggta ccgcgggccc 901 atcgctcgag agacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat 961 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa 1021 catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg 1081 gctcgtataa tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag 1141 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 1201 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 1261 tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 1321 ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag 1381 acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa 1441 ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 1501 tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 1561 ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 1621 acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 1681 aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct 1741 tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg caggcggggc 1801 gtaatctaga ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga 1861 tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat 1921 gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca 1981 tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc 2041 tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta 2101 ttgaaatgac cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt 2161 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt 2221 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctact gtcagataaa 2281 gtcccccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc 2341 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac 2401 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc 2461 tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca 2521 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt 2581 ttacgtttct cgttcagctt tcttgtacaa agtggctcga gcagggatcc gcccctctcc 2641 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 2701 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 2761 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 2821 tctgttgaat gtcgtgaagg aagcgtagga attcgccagc acagtggtcg accctgtgga 2881 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2941 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 3001 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 3061 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 3121 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 3181 gaggcttttt tggaggccta ggcttttgca aaaagcttac catgaccgag tacaagccca 3241 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 3301 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 3361 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 3421 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 3481 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 3541 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 3601 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 3661 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 3721 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 3781 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc 3841 agcgcccgac cgaaaggagc gcacgacccc atgcatcgat aaaataaaag attttattta 3901 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 3961 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa 4021 ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 4081 cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat 4141 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 4201 ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 4261 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 4321 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc 4381 cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 4441 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 4501 ggggtctttc acatgcagca tgtatcaaaa ttaatttggt tttttttctt aagtatttac 4561 attaaatggc catagttgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4621 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4681 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4741 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4801 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4861 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4921 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4981 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 5041 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 5101 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 5161 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 5221 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 5281 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5341 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5401 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5461 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt gcggccgcaa 5521 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 5581 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 5641 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 5701 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 5761 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 5821 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 5881 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 5941 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 6001 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 6061 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 6121 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 6181 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 6241 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 6301 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 6361 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 6421 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 6481 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6541 agtgccacct gcagcctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 6601 ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg 6661 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtccagcc 6721 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac 6781 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg 6841 ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg 6901 actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 6961 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 7021 tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 7081 ccgggaggca agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 7141 tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 7201 tggtggaact gacgagttct gaacacccgg ccgcaaccct gggagacgtc ccagggactt 7261 tgggggccgt ttttgtggcc cgacctgagg aagggagtcg atgtggaatc cgaccccgtc 7321 aggatatgtg gttctggtag gagacgagaa cctaaaacag ttcccgcctc cgtctgaatt 7381 tttgctttcg gtttggaacc gaagccgcgc gtcttgtctg ctgcagcgct gcagcatcgt 7441 tctgtgttgt ctctgtctga ctgtgtttct gtatttgtct gaaaattagg gccagactgt 7501 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 7561 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 7621 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 7681 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 7741 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 7801 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 7861 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccggcc ggatcccagt 7921 gtggtggtac taatagtaat caattacggg gtcattagtt ca //