LOCUS Exported 8401 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentivirus destination vector for stable expression of mCherry-tagged kinetochore marker CENP-A. ACCESSION . VERSION . KEYWORDS pLenti6-CENP-A-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8401) AUTHORS Pemble H, Kumar P, van Haren J, Wittmann T TITLE GSK3-mediated CLASP2 phosphorylation modulates kinetochore dynamics. JOURNAL J Cell Sci. 2017 Apr 15;130(8):1404-1412. doi: 10.1242/jcs.194662. Epub 2017 Feb 23. PUBMED 28232523 REFERENCE 2 (bases 1 to 8401) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8401 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind complement(98..122) /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(218..242) /label=LNCX /note="Human CMV promoter, forward primer" promoter complement(239..442) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(268..288) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 443..746 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" CDS complement(1091..1135) /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 1320..1553 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 2046..2171 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" LTR 2218..2398 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" promoter complement(2399..2625) /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" primer_bind complement(2653..2673) /label=T3 /note="T3 promoter, forward primer" promoter complement(2653..2671) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2692..2708) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2692..2708) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2705..2727) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2716..2732 /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(2740..2770) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2785..2806 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2923..2940) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3094..3682) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3174..3193) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3853..4713) /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 4476..4495 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4714..4818) /gene="bla" /label=AmpR promoter rep_origin complement(4844..5299) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4981..5002) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5193..5212 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5426..5448 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5440..5457 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5441..5457 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 5467..5486 /label=T7 /note="T7 promoter, forward primer" promoter 5467..5485 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" rep_origin 5506..5641 /label=SV40 ori /note="SV40 origin of replication" polyA_signal 5681..5802 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5692..5711) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5746..5765 /label=SV40pA-R /note="SV40 polyA, reverse primer" LTR 5874..6107 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" CDS complement(6194..6592) /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" promoter complement(6611..6658) /label=EM7 promoter /note="synthetic bacterial promoter " promoter complement(6706..7035) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6720..6855 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7020..7040 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" CDS complement(7077..7118) /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" protein_bind 7171..7195 /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS complement(7216..7926) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(7494..7513) /label=mCherry-F /note="mCherry, forward primer" primer_bind 7763..7781 /label=mCherry-R /note="mCherry, reverse primer" regulatory 7923..7932 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(7957..8046) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" ORIGIN 1 gggccggggg tcggggtcgg gctcgggctg cgcctcctcg gggcctcggg ctttcggctc 61 cggcggcgcg ggcccatggt gaagggggcg gccgcggagc ctgctttttt gtacaaactt 121 gttgatatct gcagaattcc accacactgg actagtggat cctctagagt cggtgtcttc 181 tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc actaaacgag 241 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg 301 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga 361 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga 421 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc 481 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt 541 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg 601 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta 661 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg 721 ggccatttac cgtaagttat gtaacgcgga actcccaagc ttatcgcaga cccagtcgct 781 agcgttaacg tcgacatcgc agacccagtc gctagcgtta acgtcgacat cgataccgtc 841 gagatccgtt cactaatcga atggatctgt ctctgtctct ctctccacct tcttcttcta 901 ttccttcggg cctgtcgggt cccctcgggg ttgggaggtg ggtctgaaac gataatggtg 961 aatatccctg cctaactcta ttcactatag aaagtacagc aaaaactatt cttaaaccta 1021 ccaagcctcc tactatcatt atgaataatt ttatatacca cagccaattt gttatgttaa 1081 accaattcca caaacttgcc catttatcta attccaataa ttcttgttca ttcttttctt 1141 gctggttttg cgattcttca attaaggagt gtattaagct tgtgtaattg ttaatttctc 1201 tgtcccactc catccaggtc gtgtgattcc aaatctgttc cagagattta ttactccaac 1261 tagcattcca aggcacagca gtggtgcaaa tgagttttcc agagcaaccc caaatcccca 1321 ggagctgttg atcctttagg tatctttcca cagccaggat tcttgcctgg agctgcttga 1381 tgccccagac tgtgagttgc aacagatgct gttgcgcctc aatagccctc agcaaattgt 1441 tctgctgctg cactatacca gacaataatt gtctggcctg taccgtcagc gtcattgacg 1501 ctgcgcccat agtgcttcct gctgctccca agaacccaag gaacaaagct cctattccca 1561 ctgctctttt ttctctctgc accactcttc tctttgcctt ggtgggtgct actcctaatg 1621 gttcaatttt tactacttta tatttatata attcacttct ccaattgtcc ctcatatctc 1681 ctcctccagg tctgaagatc agcggccgct tgctgtgcgg tggtcttact tttgttttgc 1741 tcttcctcta tcttgtctaa agcttccttg gtgtctttta tctctatcct ttgatgcaca 1801 caatagaggg ttgctactgt attatataat gatctaagtt cttctgatcc tgtctgaagg 1861 gatggttgta gctgtcccag tatttgtcta cagccttctg atgtttctaa caggccagga 1921 ttaactgcga atcgttctag ctccctgctt gcccatacta tatgttttaa tttatatttt 1981 ttctttcccc ctggccttaa ccgaattttt tcccatcgcg atctaattct cccccgctta 2041 atactgacgc tctcgcaccc atctctctcc ttctagcctc cgctagtcaa aatttttggc 2101 gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg 2161 cgtcgagaga gctcctctgg tttccctttc gctttcaagt ccctgttcgg gcgccactgc 2221 tagagatttt ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca 2281 acagacgggc acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg 2341 ggttccctag ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccgt 2401 ttatgtatcg agctaggcac ttaaatacaa tatctctgca atgcggcaat tcagtggttc 2461 gtccaatcca tgtcagaccc gtctgttgcc ttcctaataa ggcacgatcg taccacctta 2521 cttccaccaa tcggcatgca cggtgctttt tctctccttg taaggcatgt tgctaactca 2581 tcgttaccat gttgcaagac tacaagagta ttgcataaga ctacattaag cttgcagctc 2641 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct 2701 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 2761 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 2821 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 2881 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 2941 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 3001 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 3061 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 3121 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 3181 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 3241 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 3301 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 3361 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 3421 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 3481 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 3541 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 3601 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 3661 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 3721 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 3781 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 3841 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 3901 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 3961 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 4021 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 4081 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 4141 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 4201 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 4261 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 4321 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 4381 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 4441 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 4501 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 4561 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 4621 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 4681 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 4741 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 4801 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat 4861 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 4921 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 4981 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 5041 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 5101 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 5161 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 5221 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 5281 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 5341 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 5401 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 5461 cgcgcgtaat acgactcact atagggcgaa ttgggtacgt ccctaggcct ccaaaaaagc 5521 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa 5581 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga 5641 tgggcggagt taggggcggg atagctagag ccagacatga taagatacat tgatgagttt 5701 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 5761 attgctttat ttgtaaccat tataagctgc aataaacaag ttcctctcac tctctgatat 5821 tcatttcttt gcaagttata aatactgaat aataagatga catgaactac tactgctaga 5881 gattttccac actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag 5941 acgggcacac actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt 6001 ccctagttag ccagagagct cccaggctca gatctggtct aaccagagag acccagtaca 6061 agcaaaaagc agatcttgtc ttcgttggga gtgaattagc ccttccagtc cccccttttc 6121 ttttaaaaag tggctaagat ctacagctgc cttgtaagtc attggtctta aaggtaccga 6181 gctcgaattg tgcttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa 6241 ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga 6301 gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg 6361 cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt 6421 ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc 6481 tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag 6541 ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcttggcc atggtttagt 6601 tcctcacctt gtcgtattat actatgccga tatactatgc cgatgattaa ttgtcaacac 6661 gtgctgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag 6721 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc 6781 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg 6841 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg 6901 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga 6961 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca 7021 ccctaactga cacacattcc acagaattaa ttccaaactc attactaacc ggtacgcgta 7081 gaatcgagac cgaggagagg gttagggata ggcttacctt cgaaccgcgg gccctctaga 7141 ctcgagcggc cgccactgtg ctggatatca accactttgt acaagaaagc tgggtcggcg 7201 cgcccaccct tgcgcttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc 7261 ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt 7321 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt 7381 gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt 7441 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt 7501 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa 7561 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt 7621 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc 7681 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca 7741 ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta 7801 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat 7861 gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct 7921 caccatggtg gcgaccggtg cgcggccgca ctgagcagcg taatctggaa cgtcatatgg 7981 ataggatcct gcatagtccg ggacgtcata gggatagccc gcatagtcag gaacatcgta 8041 tgggtaaaag atgcggccgc cgagtccctc ctcaaggccc cggatcctcc gggccagttg 8101 cacatccttt gggaagagag taactcggcc tgcatgtaag gtgaggagat aggcgtcctc 8161 aaagagatga actagaaatg cttctgctgc ctcttgtagg gccaataggg cctgggcttg 8221 ccaattgaag tccacaccac gagtgaattt aacacatatt tctcttgcca ggcggctgaa 8281 gggcagcttc cttatcaaga ggtgtgtgct cttctgaagc tttcggatct cctttagcca 8341 accttgtctc cgccgactgt gttgatggga ggaagcgcct aaggaggggc cccgccggga 8401 g //