LOCUS pcDNA5-FRT-TO-FL 5876 bp DNA circular 12-JUL-2018 FEATURES Location/Qualifiers source 1..5876 /dnas_title="pcDNA5-FRT-TO-FLAG-SHLD3 for Addgene" /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 235..614 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" protein_bind 820..838 /dnas_title="tetO" /gene="tetO" /bound_moiety="tetracycline repressor TetR" /note="tet operator" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /dnas_title="tetO" /gene="tetO" /bound_moiety="tetracycline repressor TetR" /note="tet operator" /note="bacterial operator O2 for the tetR and tetA genes" CDS 993..1016 /dnas_title="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /note="FLAG" /translation="DYKDDDDK" polyA_signal 1834..2058 /note="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" protein_bind 2342..2389 /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FRT" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 2397..3419 /dnas_title="HygR" /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /note="HygR" /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGDFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYH WQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSE AMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQS LVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPST RPRAKE" polyA_signal 3549..3670 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" primer_bind complement(3719..3735) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 3743..3759 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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(3767..3797) /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 3812..3833 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4121..4709) /dnas_title="ori" /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4880..5740) /dnas_title="AmpR" /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" promoter complement(5741..5845) /dnas_title="AmpR promoter" /gene="bla" /note="AmpR promoter" CDS 1041..1787 /note="human SHLD3" /translation="TTEVILHYRPCESDPTQLPKIAEKAIQDFPTRPLSRFIPWFPYD GSKLPLRPKRSPPVISEEAAEDVKQYLTISEHDAKSHSYDCTVDLLEFQPSLKKQHLT WSHTLKEQTNSGNLGKQSEKGKQHKRRSWSISLPSNNCTKNVSPLSKKLQDSLKALNL HSLYRARWTIEHTICNSQTLEDIWTKLNQIIRHNELPSCNATIQRHLGQIWVFCDIMY CEYVGSLLKGRLALTGKINLFVHKYGVIFSM" CDS 990..1787 /note="FLAG-SHLD3" /translation="MDYKDDDDKAASLNGAPTTEVILHYRPCESDPTQLPKIAEKAIQ DFPTRPLSRFIPWFPYDGSKLPLRPKRSPPVISEEAAEDVKQYLTISEHDAKSHSYDC TVDLLEFQPSLKKQHLTWSHTLKEQTNSGNLGKQSEKGKQHKRRSWSISLPSNNCTKN VSPLSKKLQDSLKALNLHSLYRARWTIEHTICNSQTLEDIWTKLNQIIRHNELPSCNA TIQRHLGQIWVFCDIMYCEYVGSLLKGRLALTGKINLFVHKYGVIFSM" 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 ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtacca tggactacaa agacgatgac gataaagccg 1021 ccagtttaaa cggcgcgcca ACTACAGAAG TAATATTACA TTATCGACCA TGTGAGAGTG 1081 ATCCCACACA ACTGCCAAAA ATTGCAGAAA AAGCAATTCA AGACTTTCCT ACTCGTCCGC 1141 TATCAAGATT TATACCTTGG TTTCCATATG ATGGGTCCAA GCTTCCACTC AGACCTAAAA 1201 GATCACCACC TGTGATTTCT GAAGAGGCAG CTGAAGATGT GAAACAGTAC TTAACCATTT 1261 CAGAACATGA TGCTAAGTCA CACAGTTATG ATTGCACAGT AGATCTATTG GAGTTTCAAC 1321 CTAGCTTGAA AAAGCAGCAT TTAACCTGGT CACACACACT GAAGGAACAG ACTAATTCTG 1381 GAAATCTGGG TAAACAATCA GAAAAGGGAA AACAGCACAA GAGGAGATCT TGGAGTATTT 1441 CCCTTCCCAG CAATAATTGT ACTAAAAACG TTTCTCCTTT GTCTAAAAAA TTGCAAGATA 1501 GTTTAAAGGC ACTAAATTTA CACTCACTTT ATAGAGCAAG ATGGACAATA GAACACACTA 1561 TTTGTAACAG CCAAACTCTG GAAGACATTT GGACAAAACT CAATCAAATT ATTAGGCACA 1621 ATGAACTTCC ATCTTGTAAT GCTACAATTC AGAGGCATTT AGGCCAAATA TGGGTGTTCT 1681 GTGATATTAT GTATTGTGAA TATGTGGGAA GTCTTCTTAA AGGAAGATTA GCTCTTACTG 1741 GAAAAATTAA TTTATTTGTG CATAAATATG GTGTTATTTT TAGTATGTAG ctcgagtcta 1801 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 1861 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 1921 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 1981 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 2041 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc 2101 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 2161 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 2221 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 2281 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct 2341 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa 2401 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct 2461 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag 2521 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg 2581 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggatt 2641 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc 2701 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg 2761 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc 2821 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc 2881 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc 2941 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 3001 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 3061 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg 3121 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc 3181 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt 3241 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga 3301 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 3361 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc 3421 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 3481 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 3541 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 3601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 3661 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt 3721 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 3781 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 3841 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 3901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 3961 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 4021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 4141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 4321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4561 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4861 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4921 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 4981 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 5041 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 5101 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 5161 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 5221 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 5281 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 5341 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 5401 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5461 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5521 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5581 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5641 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5701 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5761 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5821 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc //