LOCUS Exported 7349 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor TCFL5 with GFP. ACCESSION . VERSION . KEYWORDS pTCFL5-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7349) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7349) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7349 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(34..50) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(34..50) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(47..69) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 58..74 /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(82..112) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 127..148 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(265..282) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(436..1024) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(516..535) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1195..2055) /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 1818..1837 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2056..2160) /gene="bla" /label=AmpR promoter primer_bind 2228..2246 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2284..2306) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2406..2425 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2619..2641 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2633..2650 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2634..2650 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 3877..3897 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3910..3954 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3994..4017 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 4045..4764 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK EFVTAAGITLGMDELYK" primer_bind complement(4090..4111) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4351..4370) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4802..5387 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4968..4985) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5195..5214 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5469..6272 /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5532..5551) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6142..6161 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 61 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 121 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 181 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 241 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 301 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 361 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 421 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 481 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 541 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 601 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 661 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 721 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 781 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 841 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 901 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 961 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 1021 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 1081 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 1141 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 1201 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 1261 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 1321 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 1381 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 1441 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 1501 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 1561 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 1621 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 1681 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 1741 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 1801 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 1861 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 1921 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 1981 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 2041 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 2101 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 2161 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 2221 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga 2281 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 2341 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa 2401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca 2461 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg 2521 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 2581 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 2641 gacggccagt gaattcgagc tcggtacccc cccttccttt cttcctcacc gttcatttgt 2701 tgaaaaggga atgatttgtc ccgtagacgt ttccacattc tgcccgtggt ggttggcgcc 2761 tgtcatcggg ccgtcgcctg tgcccctctc ttcctctcag tctccttcgg gaggttgaga 2821 ctgaccccac cattataatt gaggcctttt gtaaaagaca ggtggtgtga caggagcaag 2881 ctcagcccac ccccaccctg ctggcagggt ctccccaagg acagagccat ctcagcgagc 2941 agcacctcca ccttgctggc cttggaaccc tgagtgtgtg gcagtgcaga gggcaaggca 3001 gtcgccctgg ggtgggggta gatgcccctg acttgttaat tgttgaaact ttttaaaaat 3061 ttattttttt gagacggagt ttcactcttg ttgcccagac tggagtgcaa tggcatgatc 3121 tcggctcact gcaacctcca cctcccaggc tcaagagatt gtcctgcctc agcctcccaa 3181 gtagctgggg tcacaagatg cttgccacca cgcccagcta atttttgtat ttttagtaga 3241 gatggggttt caccatgtta atcaggctgg tcctgaactc ctgacgtcag gtgatccacc 3301 cacctcggcc tcccaaagtg ctgggattat aggcgtgagc cgccacgccc ggcctgaaac 3361 gttttaatat atacactcta ccttctctca cacgctgggt ttacaggttt attagctagg 3421 gcccattagc ggttgaaacg ttcatgtcat gtatatttta ccacaataaa atcaatacag 3481 tgtggccaga cattctgagt attggaaaaa caaaagtaca tgtacagtgt aactgctatt 3541 tgaaaaaaaa aagttaaaat ctgtttatta tataaatccc cttgtgaggt aagacacaag 3601 tttgtgaatg acaccttaaa acagcgtatt ctaaagtcat ttacttagct cttaatttga 3661 accttttgca ctttggaaga cagcagtttt catgtcagtt aattcagtat gttaaccgtc 3721 acttcttcac tgcaggaatt tgagagcgta ttttgcggta aaactggccg aaggctaaag 3781 ctgaccagac cggactcctt ggtgacctgt cctgcacagg ggagtttaca gagcagcccc 3841 tcgatggaga tcaaggggat tccaactact gcaagcgaga atctttattt tcagggcgcc 3901 gccaaattca aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga cagcggaggt 3961 ggaggttcga gcggtccctc gggttcgtcg agcctggaag ttctgttcca ggggcccctg 4021 tcgtcgagcg gtccctcggg ttcgatggtg agcaaaggcg aggagctgtt caccggggtg 4081 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 4141 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 4201 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 4261 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 4321 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 4381 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 4441 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 4501 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 4561 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 4621 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 4681 aacgagaagc gcgatcacat ggtccttaag gagttcgtga ccgccgccgg gatcactctc 4741 ggcatggacg agctgtacaa gtaatgatct agagtcgagt taattaagaa ttccgccccc 4801 ccccctctcc ctcccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 4861 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 4921 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 4981 aatgcagggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 5041 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 5101 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 5161 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 5221 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 5281 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 5341 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaacctgt tacattgcac 5401 aagataaaaa tatatcatca cgaacagtaa aactgtctgc ttacataaac agtaatacaa 5461 ggggtgttat gggatcggcc attgaacaag atggattgca cgcaggttct ccggccgctt 5521 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 5581 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 5641 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 5701 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 5761 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 5821 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 5881 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 5941 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 6001 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 6061 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 6121 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 6181 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 6241 ccttctatcg ccttcttgac gagttcttct gagtctcgga ctgaacagga atcctcgggg 6301 ggtgaacagc cattccttcg tgacctgtgc aacgccttct gcaaccctgg agctctgctc 6361 ggctagtctg actcgaaaag ggcgtgactc aagctgacgg gactccagta gggactttga 6421 gagcacattt tgtaaaaata tttatctaga cgcaaatgct tatccatgaa tgtcctctta 6481 gaccatttgg ggatgaagcc atcttaataa ttagtaataa ttaattagta ataattagta 6541 agcattttct caatgctctg attccatcat gttttcttaa catgataact taaaaaattg 6601 acatcctttg tactttcttt aatcttaaaa agtacacggc tttttactta tttacctttt 6661 aaaattgccc ctttagcaat tggaacaagt taaattgtta actaaaaaca gtttggaaat 6721 tttatttcat tcgttatatc acaccccctt gtcatgactc tgagtcacgt gctgctgtat 6781 tgcaacgtgc aggagcattt taaacctgtg tgctaaaaat tttccagata cttgctttaa 6841 agctactttt gtccacaaat gaaatactgt cacagtagac gcttaaatgc cacgttttca 6901 taccaagagt cattcattac ttcatgtgtc acaaactgtg gtgtttggaa ttgggttttt 6961 caatgagtgg ctttacttat cacaacaggt aatagcaata gatgttagtg caatacaaag 7021 tcaccctcaa taaatactgt taattggaga tgagatgtga gtttgtacac aaaacatcag 7081 actagacctt tgtatgggag agaatttact gtacattaaa ttctttattt tttgttactc 7141 tgtagccagt cattaatctg aagatttaat atatcatttt attgggatga gatcatagtc 7201 tttacacaaa tgctatgcaa acaagttact gaatattttt cacctcgtgg agttgtacac 7261 aaccttttat atatacacac cctaccttct cgcaaatgct gggcttacag gtttattagc 7321 tagggccttt tgaggtatgc tgtcaggcg //