LOCUS Exported 7975 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor ATF1 with GFP. ACCESSION . VERSION . KEYWORDS pATF1-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7975) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7975) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7975 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(70..89) /label=T7 /note="T7 promoter, forward primer" promoter complement(71..89) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(96..113) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(96..112) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(105..127) /label=M13/pUC Forward /note="In lacZ gene" CDS 250..552 /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 431..450 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 901..1695 /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" regulatory 1895..1904 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1901..2275 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 2413..3001 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2902..2921 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3155..3172 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3289..3310 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3325..3355 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3363..3379 /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)." primer_bind 3368..3390 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3387..3403 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3387..3403 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 3421..3439 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3421..3438 /label=SP6 /note="SP6 promoter, forward primer" CDS 4422..4442 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 4455..4499 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 4539..4562 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 4590..5309 /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(4635..4656) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4896..4915) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 5347..5932 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(5513..5530) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5740..5759 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 6014..6817 /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" ORIGIN 1 aagggcgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg 61 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt 121 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 181 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta 241 tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg 301 gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt 361 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat 421 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa 481 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc 541 tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc 601 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct 661 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca 721 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg 781 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca 841 aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc 901 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 1021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 1081 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 1141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 1201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 1261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 1321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 1381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 1441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 1501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 1561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 1621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 1681 gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg 1741 catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac 1801 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 1861 ctgataaatg cttcaataat agcacgtgag gagggccacc atggccaagt tgaccagtgc 1921 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct 1981 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 2041 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 2101 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 2161 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 2221 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt 2281 gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 2341 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 2401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 2461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 2521 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 2581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 2641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 2701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 3001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 3061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 3121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 3181 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 3241 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 3301 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 3361 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 3421 atttaggtga cactatagaa tactcaagct atgcatcaag cttggtaccg agctcggatc 3481 cactagtaac ggccgccagt gtgctggaat tcgccctttg gccaatactt ccgatttcta 3541 tatgtatata aacttacata tgacttcaca gaaatgtttt aagaggacat gcacagaact 3601 gagtatttgc tcaagcattg tttgtaattt gaacttaaat aaaagaagtc atgtgtcaga 3661 agttaaccag aggacccata atttcactct ttggtattta ttcaagagaa atgaaacctt 3721 tctacaaaaa gactggcaca aaaacatgcc tatgcagcta accctagaaa catcgcaaga 3781 gactggatac acaaatggtg gttatattca tacagtgaac tatgtccagc agtgtgaagg 3841 aacaaacccc catgcagcaa catggatgaa cctcagatgg gctgagtgaa gggacaccag 3901 agtacatagt gtatgacact cttacatgag gttctagaat aggaaaaaaa ctaatctctg 3961 gtgataaaaa tcaaaacgat agttgcttgg aagggggctg accaggaagg ggcatgaggg 4021 aactttctgg gacagtggga atgttctgtt gtcttgacag aggtgtgggt tatacaggta 4081 tacacaattg ccaaaactga ctgaattcta cacttaagat ctatgtattc tctgtgtgta 4141 gatgatactt taaagagaaa aaaaggtgac cacggaaaat tactgaagaa catttaccat 4201 ttaaagaatg tgaagttttt tctaacattg tttttttaat gctcatattt agagaagctg 4261 ctcgagaatg tcgcagaaag aagaaagaat atgtgaaatg cctggaaaac cgagttgcag 4321 tcctggaaaa tcaaaataaa actctaatag aagagttaaa aactttgaag gatctttatt 4381 ccaataaaag tgttgattat gatattccaa ctactgcaag cgagaatctt tattttcagg 4441 gcgccgccaa attcaaagaa accgctgctg ctaaattcga acgccagcac atggacagcg 4501 gaggtggagg ttcgagcggt ccctcgggtt cgtcgagcct ggaagttctg ttccaggggc 4561 ccctgtcgtc gagcggtccc tcgggttcga tggtgagcaa aggcgaggag ctgttcaccg 4621 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 4681 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 4741 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 4801 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 4861 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 4921 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 4981 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 5041 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 5101 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 5161 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 5221 accccaacga gaagcgcgat cacatggtcc ttaaggagtt cgtgaccgcc gccgggatca 5281 ctctcggcat ggacgagctg tacaagtaat gatctagagt cgagttaatt aagaattccg 5341 cccccccccc tctccctccc cccccctaac gttactggcc gaagccgctt ggaataaggc 5401 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg 5461 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc 5521 aaaggaatgc agggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga 5581 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg 5641 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag 5701 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc 5761 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct 5821 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac 5881 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa cctgttacat 5941 tgcacaagat aaaaatatat catcacgaac agtaaaactg tctgcttaca taaacagtaa 6001 tacaaggggt gttatgggat cggccattga acaagatgga ttgcacgcag gttctccggc 6061 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 6121 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 6181 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac 6241 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 6301 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 6361 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 6421 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 6481 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 6541 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 6601 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 6661 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 6721 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 6781 catcgccttc tatcgccttc ttgacgagtt cttctgaatt cctaagaaag aaaatatttt 6841 tgtggacatg cataaaaatt aaatggattt cctagtggag ttttataaat taaaaggtca 6901 aaactgaagc tttttattta ggcttttcca aatcaaggat aaatatctta cgcacgatat 6961 ctagtgacag aggagaaagt ggaaaatgac ctcaaggaag ctacgggcac aactggaagc 7021 tttgtagaaa ttaaacatat tcaaggagca agaaatgaac tttcagcagt ctaaattttc 7081 taaataacca atagttgcca atctaaaatg gcagagaaga tgaaatttga taaactgaat 7141 tttttttaaa aatccattta ccctacaggt ttgcatttgt ttgctgaaat ttaccttttt 7201 ttagttatat atatgtgtgt gtgtgtgtgt aatttctgcc aataaattct aaattacaaa 7261 ggtaagagaa aacctagtac attactaaat atataaagta tatgttctga ttatgtatac 7321 ttgttctagt gtcaagtctt tttaagtggg tttttaaaag tttgttattg gacttgaatg 7381 gatttttgag actaggttaa ttatttttga ggtcttatcc taaaaggcat ctaaggtaca 7441 tgaatggagt atggtgattt tataacattt tttatcagaa tggaaaaaga actgtttaaa 7501 agtttgatac ttttaaatag ttggtttttt tgcttactct ggtaatgatt ttctacaaat 7561 acataataaa ttgttttttt gagtctatat tctgtatgca gttgaatatc cattacttat 7621 tctgctgtgc tttaatagaa tggaatgttt acaggccctt aaaatattat ttttaaaaaa 7681 ccttctgaag atacatacca aagtttttcc aagaagattt tataatcaat ttaataatgt 7741 aaggtttatc agattctata atagtatagt tattaaggca attttatgtt agagactatt 7801 ttgtaatgta gtgagtggta cctttataag aaaagtgact gccaatatat ttttatagct 7861 gatctttata aattctaatg ttgagttttt aatgattatt ttaaatgttt atatagttta 7921 gtaaaatttg catctcaaag tatcattttt atattatggg acgttttcag attgg //