LOCUS Exported 7065 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor E2F3 with GFP. ACCESSION . VERSION . KEYWORDS pE2F3-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7065) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7065) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7065 /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 3733..3753 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3766..3810 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3850..3873 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 3901..4620 /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(3946..3967) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4207..4226) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4658..5243 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4824..4841) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5051..5070 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5325..6128 /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(5388..5407) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5998..6017 /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 tagaaggaca 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 caggagttcg aggttagagt gagctatgct 2701 tgcctgtgaa tagccactac actccagcgt agacaacata gcaagacgcc gtctcttaaa 2761 aaaaaaagtt cttttttcag ttggtgagtt tctactagga agccaacttt tcagtatgta 2821 tatattgttt tgttttgatt ttcttaaaac aatgactaga tagcagagag aagtttttga 2881 acttgttatc ctctcatcat tgaagttatt tgcattcttg tttattgaca gtagttattt 2941 gaaaaatgag tgagtagact agggccaaag aaagatggcc aggcatggtg gctcacacct 3001 gtaatttcag cactttggga ggccaaggaa gacagatcac ttgagcccag gagttcgaga 3061 ccagactggg caacatggca aaaccctgtc tctacaaaaa aagattaacc aggcatggtg 3121 gctcacacct ggagtcccag ctacttggga ggctgaggtg ggagggtccc ttgaacccag 3181 aagttcaaga ctgcagtgag tcatgatcac accactgcac tccagccaag agaataagac 3241 cctgtctcaa aaattaaaag ggggacgggg gtggcaagga acttttttgt acctgttagc 3301 ataaaaggtg atctgcatca taaagtcgtc tcattgtcat tatttgcgga aggtacatgc 3361 ttttttctaa cagcaacata tacattcttc cagaaaaatt catttgattt ttctaactta 3421 ttttttgttt ccatcaatgt tttcttttca gacttggctt caaccaactc aggacatagc 3481 gattgctcag tttctatggg aaacctttct cctctggcct ccccagccaa cctcttacag 3541 cagactgagg accaaattcc ttccaaccta gaaggaccgt ttgtgaactt actgcctccc 3601 ctgctgcaag aggactatct cctgagcctc ggggaggagg aaggcatcag cgatctcttc 3661 gatgcttacg atttggaaaa gctcccactg gtggaagact tcatgtgtag tgggattcca 3721 actactgcaa gcgagaatct ttattttcag ggcgccgcca aattcaaaga aaccgctgct 3781 gctaaattcg aacgccagca catggacagc ggaggtggag gttcgagcgg tccctcgggt 3841 tcgtcgagcc tggaagttct gttccagggg cccctgtcgt cgagcggtcc ctcgggttcg 3901 atggtgagca aaggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 3961 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 4021 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 4081 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 4141 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 4201 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 4261 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 4321 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 4381 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 4441 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 4501 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 4561 cttaaggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 4621 tgatctagag tcgagttaat taagaattcc gccccccccc ctctccctcc ccccccctaa 4681 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc 4741 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 4801 gagcattcct aggggtcttt cccctctcgc caaaggaatg cagggtctgt tgaatgtcgt 4861 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 4921 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 4981 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 5041 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 5101 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 5161 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 5221 acgatgataa tatggccaca acctgttaca ttgcacaaga taaaaatata tcatcacgaa 5281 cagtaaaact gtctgcttac ataaacagta atacaagggg tgttatggga tcggccattg 5341 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 5401 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 5461 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 5521 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 5581 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 5641 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 5701 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 5761 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 5821 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 5881 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 5941 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 6001 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 6061 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 6121 tcttctgagt ctgattatgc ttcgtgtgaa ctctccttaa aaaccgatat ttttttatca 6181 tggaaccaga acatctgtca tgcagtgttg tcccttccta ccttcttcct ccaagagagt 6241 atcatgaagt aaactacaaa cttcagaaga aagctgacat tttaatgaat tttttaaaaa 6301 attaataaac aaattgtcta aacgcacagt tgcaggctcc cttgggaaag ccctgctttg 6361 ctccaggctc caagatctcc tggctaagtc agcaagtgag aaaatgtgca atcaggtgtc 6421 tctcaccccg aattgtcctt cctccttcct ccccggattg gcttgctgtg cctgacggat 6481 gggctgtaga atggggtctg gccacctggc ctgctgggaa acagcaatct tccttaatag 6541 catttcaagc cgtgccttct ccgcagaatg catgtctttg aggtctgcta atatggaatg 6601 gaactgcagc aaatgcaaac ttgaagtcat gcaaaagtat gaaatggatt tcttcagctc 6661 ttcttaggaa tatttaaatt actgtcataa ttcagtttaa gctatgaact gtgtgtccca 6721 gtaggaggtc aagaaaacct ccacagcctt ctggatgaag aacctgtttt caaatatact 6781 tgttgcagat acagaagact agtagagttc tgccactcta agctgttgtg gattttcctg 6841 tctccatgaa ccactcccat tcccccgtcc ccaatgtgtt tgtgagtttc cagttgattt 6901 gtagcaaatg cctacttagt tctttgtgga ttgttctaga cttttaattt ttttagctgc 6961 catttaagca ttcctgtggc acccatcacc atttcaattt aattgtttac tttgaagcgg 7021 tttttgcaaa ttcatattac ttaagcagag ggagagaacc tctac //