LOCUS Exported 9086 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of IFT88 fused with N terminal TagRFP.T tag. ACCESSION . VERSION . KEYWORDS pEF5-FRT-TagRFP-T-IFT88 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9086) AUTHORS Ye F, Breslow DK, Koslover EF, Spakowitz AJ, Nelson WJ, Nachury MV TITLE Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors. JOURNAL Elife. 2013 Aug 6;2:e00654. doi: 10.7554/eLife.00654. Print 2013. PUBMED 23930224 REFERENCE 2 (bases 1 to 9086) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9086 /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" promoter 353..1531 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 584..1522 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1479..1499 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind 1548..1567 /label=T7 /note="T7 promoter, forward primer" promoter 1548..1566 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1612..2343 /codon_start=1 /product="photostable variant of TagRFP (Shaner et al., 2008)" /label=TagRFP-T /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV ARYCDLPSKLGHKLNGMDELYK" protein_bind 2380..2404 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(4883..4907) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 4960..5001 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" primer_bind complement(5039..5056) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5045..5269 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5402..5421) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 5552..5599 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 5607..6629 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 6759..6880 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6796..6815) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6850..6869 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6929..6945) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6929..6945) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6942..6964) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6953..6969 /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(6977..7007) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7022..7043 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7160..7177) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7331..7919) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7411..7430) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8090..8950) /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 8713..8732 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8951..9055) /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 taggcttttg caaaaagctt tgcaaagatg gataaagttt taaacagaga 301 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgcctcgt gaggctccgg 361 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt 421 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt 481 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc 541 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg 601 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac 661 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 721 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg 781 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa 841 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag 901 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 961 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 1021 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc 1081 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 1141 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 1201 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc 1261 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc 1321 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 1381 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt 1441 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca 1501 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtactaa tacgactcac 1561 tatagggaga cccaagctgg ctaggtaagc ttggtaccga gctcggatcc gatggtgtct 1621 aagggcgaag agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg 1681 aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag 1741 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct 1801 accagcttca tgtacggcag cagaaccttc atcaaccaca cccagggcat ccccgatttc 1861 tttaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg 1921 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc 1981 aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc 2041 tgggaggcca acaccgagat gctgtacccc gctgacggcg gcctggaagg cagaaccgac 2101 atggccctga agctcgtggg cgggggccac ctgatctgca acttcaagac cacatacaga 2161 tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga ccacagactg 2221 gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc 2281 agatactgcg acctccctag caaactgggg cacaaactta atggcatgga cgagctgtac 2341 aagggactag tccagtgtgg tggaattctg cagatatcaa caagtttgta caaaaaagca 2401 ggcaccatgg aaaatgttca tctggcacca gaaacagatg aggacgacct ttactctggt 2461 ttcaacgact acaacccagc ctatgatact gaggagttgg agaatgacac gggctttcag 2521 caagcagtga gaaccagtca tggcagaaga cctcccgtaa ctgctaaaat accaagcaca 2581 gcagtcagca ggcccacagc tactggatat gggtccaaga catccctgac atcatcaatg 2641 ggaagaccga tgacagggac aattcaggat ggagttgctc gaccgatgac agcagtgaga 2701 gcagctggct tttccaaggc agctttgcga ggctctgcat ttgaccccct tggtcagtcc 2761 aggggccctg ctcctccttt ggaagccaaa aatgaagaca gtccagaaga aaaaattaga 2821 cagttggaga agaaagtaaa tgagttggtg gaagaaagct gtattgccaa cagttgtgga 2881 gacttaaaac tggccttaga gaaggcaaaa gatgcaggaa gaaaagagag agtcctggtg 2941 agacaacgag agcaagtcac aagtccagaa aatatcaact tggacttaac ctactccgtt 3001 cttttcaact tggccagtca gtattctgct aatgaaatgt atgcagaagc actgaacaca 3061 taccaagtta tagtgaaaaa caagatgttt agcaatgcag gaagactgaa agtgaatatg 3121 ggaaatattc atttaaagca gagaaattat tccaaagcca ttaagttcta ccgaatggcc 3181 ttagatcaga tcccaagtgt ccataaagaa atgaggatca aaataatgca gaacattgga 3241 atcacattta taaaaactgg tcagtactca gatgccatca actcatttga gcacatcatg 3301 agcatggccc cgagcctgaa ggcaggcttc aacctcatcc ttagttgttt tgccattgga 3361 gatcgggaga aaatgaagaa ggcattccaa aaattaattg ctgttccgtt agaaattgat 3421 gaagatgata aatatatctc cccaagtgat gatccccata ctaacttgct gattgaagct 3481 ataaaaaatg accaccttag gcaaatggaa cgtgaaagga aagccatggc agaaaaatac 3541 atcatgacag ctgcaaaact cattgctcct gtgatcgaag cgtcttttgc tgtgggttat 3601 aactggtgtg tggaagtagt gaaagcctct cagtatgtgg agctggccaa cgacctggag 3661 attaacaaag caattactta cttgagacaa aaggacttta accaagctgt agacacattg 3721 aaaatgtttg agaagaagga cagtagagtg aagagtgcag ctgcgaccaa cctctcgttc 3781 ctgtattatc tggaaaacga atttgcccaa gccagcagct atgcagattt agctgtgaac 3841 tcggatagat ataacccgtc agctcttact aataaaggga acacagtttt tgcaaatggt 3901 gattatgaga aggcagctga gttctataaa gaggccctga gaaacgactc ttcctgtact 3961 gaagcactgt ataacattgg cctcacctat aagaagctaa accgtctgga tgaagccttg 4021 gactccttcc tgaaactgca cgcaatcctt cggaacagcg ctcaagttct ttgccagata 4081 gcaaacatat atgagttaat ggaagatccc aatcaagcta ttgaatggct gatgcagttg 4141 atcagcgttg ttccaactga ttcccaagct ctgtctaaac tgggagagtt atacgatagt 4201 gagggagaca agtcccaggc attccaatat tactatgagt catacaggta tttcccttct 4261 aacattgaag tcattgaatg gcttggagct tattacattg atacacagtt ctgcgagaaa 4321 gccattcagt actttgaaag agcttccctt atacaaccca cacaagtgaa gtggcagctg 4381 atggtagcta gctgctttag acgaagtggt aactaccaaa aggcattaga tacttataaa 4441 gagattcaca ggaaatttcc agagaatgtt gaatgtttgc gtttcttggt tcgtctctgc 4501 acagagattg gattaaaaga agttcaagaa tatgccacaa aactcaagag gttggaaaaa 4561 atgaaagaaa tgagggaaca gcgcataaaa tcgggcagag acagcagcgg gggttctcgc 4621 agcaaacgag aggggagtgc tggcagtgac agtggccaga acaatagtgc cagtagtaag 4681 agtgaacgac tgagtgccaa gctcagagct ttgcctggga cagatgaacc ttatgaaagt 4741 agcggtaaca aagaaataga tgcctcctac gtggatccac tgggccctca gatagaaaga 4801 ccaaaaactg cagccaagaa aagaattgac gaggatgatt ttgctgatga agagttagga 4861 gatgacttgc tcccagaata gaacccagct ttcttgtaca aagtggttga tatccagcac 4921 agtggcggcc gctcgagtct agagggcccg cggttcgaag gtaagcctat ccctaaccct 4981 ctcctcggtc tcgattctac gcgtaccggt tagtaatgag tttaaacccg ctgatcagcc 5041 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 5101 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 5161 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 5221 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 5281 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc 5341 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 5401 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5461 ctaaatcggg ggtcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 5521 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc 5581 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga 5641 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag 5701 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct 5761 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc 5821 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc 5881 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc 5941 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt 6001 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg 6061 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt 6121 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc 6181 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag 6241 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct 6301 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc 6361 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc 6421 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat 6481 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa 6541 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc 6601 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg 6661 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 6721 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata 6781 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 6841 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga 6901 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 6961 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 7021 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 7081 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 7141 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 7201 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 7261 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 7321 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 7381 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 7441 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 7501 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 7561 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 7621 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 7681 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 7741 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 7801 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 7861 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 7921 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 7981 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 8041 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 8101 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 8161 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 8221 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 8281 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 8341 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 8401 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 8461 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 8521 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 8581 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 8641 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 8701 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 8761 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 8821 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 8881 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 8941 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 9001 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 9061 ttccccgaaa agtgccacct gacgtc //