LOCUS Exported 7112 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor ESR1 with GFP. ACCESSION . VERSION . KEYWORDS pESR1-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7112) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7112) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7112 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1067..1087 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1100..1144 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 1184..1207 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 1235..1954 /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(1280..1301) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1541..1560) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 1992..2577 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2158..2175) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2385..2404 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2659..3462 /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(2722..2741) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3332..3351 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4513..4529) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4513..4529) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4526..4548) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4537..4553 /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(4561..4591) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4606..4627 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4744..4761) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4915..5503) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4995..5014) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5674..6534) /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 6297..6316 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6535..6639) /gene="bla" /label=AmpR promoter primer_bind 6707..6725 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6763..6785) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6885..6904 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7098..8 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7112..17 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" ORIGIN 1 gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg gctgagcctg cagaggcttg 61 aggctgctgc cactgggtag cggtaggcct ttccgaggag gcggcatttg aagaccggag 121 gaaggttcat cccagcaagt aggaacagca agtgtaggtc ccctaagtct tgggggagct 181 tagttccttt aagggcagca caaaaatcag tgtggctccg gagagcacat taggggagag 241 aggcaggaag agcttggaga catggatgga agctggacca gttgggcctt gttgaacatg 301 gaaaggcatt tagatcgtat tctgagttaa atgggaagtg acgtgagaga tttaacaatg 361 gagcgtcttg aactgcttta ctcatttaaa atacccactc ctgcttggct gaatatctca 421 tgttgtcttt ttagaagctt tggcgatcct atttgaatgc atttaggtcc tattggaggg 481 gaataggatc tcatttgagg ccacggaggt ccatggaagt cacctgcata gcaaataccc 541 tgaaagtggc tgcagggaga gtgtgagggt gggaccgccc tggtaggagg tggaaaatga 601 aaaacacacg gccatgagtt ccagattagg gcttctgaaa gccctcagct ttcccagctc 661 ccatcctaaa gtgggtcttt aaacaggaag aaagaaagat tgctaagtgt ctttggagtt 721 cctcttcctt ccccttctag ggatttcagc actccagggg ctcgggttgg ctctaaagta 781 gtcctttctg tgtcttccca cctacagtaa caaaggcatg gagcatctgt acagcatgaa 841 gtgcaagaac gtggtgcccc tctatgacct gctgctggag atgctggacg cccaccgcct 901 acatgcgccc actagccgtg gagaggcatc cgtggaggag acggaccaaa gccacttggc 961 cactgcgggc tctacttcat cgcattccct gcaaaagtat tacatcacgg gggaggcaga 1021 gggtttccct gccacggtcg atcccgggat tccaactact gcaagcgaga atctttattt 1081 tcagggcgcc gccaaattca aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga 1141 cagcggaggt ggaggttcga gcggtccctc gggttcgtcg agcctggaag ttctgttcca 1201 ggggcccctg tcgtcgagcg gtccctcggg ttcgatggtg agcaaaggcg aggagctgtt 1261 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1321 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1381 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 1441 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1501 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1561 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1621 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1681 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 1741 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 1801 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 1861 caaagacccc aacgagaagc gcgatcacat ggtccttaag gagttcgtga ccgccgccgg 1921 gatcactctc ggcatggacg agctgtacaa gtaatgatct agagtcgagt taattaagaa 1981 ttccgccccc ccccctctcc ctcccccccc ctaacgttac tggccgaagc cgcttggaat 2041 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg 2101 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc 2161 tcgccaaagg aatgcagggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt 2221 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg 2281 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac 2341 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg 2401 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg 2461 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc 2521 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaacctgt 2581 tacattgcac aagataaaaa tatatcatca cgaacagtaa aactgtctgc ttacataaac 2641 agtaatacaa ggggtgttat gggatcggcc attgaacaag atggattgca cgcaggttct 2701 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 2761 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 2821 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 2881 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 2941 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 3001 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 3061 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 3121 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 3181 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 3241 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 3301 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 3361 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 3421 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagtcgactg agagctccct 3481 gggtcccaca cggttcagat aatccctgct gcattttacc ctcatcatgc accactttag 3541 ccaaattctg tctcctgcat acactccggc atgcatccaa caccaatggc tttctagatg 3601 agtggccatt catttgcttg ctcagttctt agtggcacat cttctgtctt ctgttgggaa 3661 cagccaaagg gattccaagg ctaaatcttt gtaacagctc tctttccccc ttgctatgtt 3721 actaagcgtg aggattcccg tagctcttca cagctgaact cagtctatgg gttggggctc 3781 agataactct gtgcatttaa gctacttgta gagacccagg cctggagagt agacattttg 3841 cctctgataa gcacttttta aatggctcta agaataagcc acagcaaaga atttaaagtg 3901 gctcctttaa ttggtgactt ggagaaggct aggtcaaggg tttattatag caccctcttg 3961 tattcctatg gcaatgcatc cttttatgaa agtggtacac cttaaagctt ttatatgact 4021 gtagcagagt atctggtgat tgtcaattca ttccccctat aggaatacaa ggggcacaca 4081 gggaaggcag atcccctagt tggcaagact attttaactc gatacactgc agattcagat 4141 gtgctgaaag ctctgcctct ggctttccgg tcatgggttc cagttaattc acgcctccca 4201 tggacctatg gagagcagca agttgatctt agttaagtct ccctatacga gggataagtt 4261 cctgattttt gtttttattt ttgtgttaca aaagaaagcc ctccctccct gaacttgcag 4321 taaggtcagc ttcaggacct gttccagtgg gcactgtact tggatcttcc cggcgtgtgt 4381 gtgccttaca caggggtgaa ctgttcactg tggtgatgca tgatgagggt aatggtagtt 4441 gaaaggagca ggggccctgg tgttgcattt agccctgtcg acctgcaggc atgcaagctt 4501 agcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4561 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4621 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4681 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4741 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4801 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4861 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4921 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4981 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 5041 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 5101 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 5161 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 5221 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 5281 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5341 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5401 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5461 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5521 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5581 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5641 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5701 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5761 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5821 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5881 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5941 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6001 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6061 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6121 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6181 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6241 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6301 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6361 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6421 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6481 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6541 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6601 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6661 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 6721 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 6781 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 6841 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat 6901 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 6961 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 7021 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 7081 ggtaacgcca gggttttccc agtcacgacg tt //