LOCUS Exported 7103 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor CLOCK with GFP. ACCESSION . VERSION . KEYWORDS pCLOCK-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7103) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7103) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7103 /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 3891..3911 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3924..3968 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 4008..4031 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 4059..4778 /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(4104..4125) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4365..4384) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4816..5401 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4982..4999) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5209..5228 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5483..6286 /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(5546..5565) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6156..6175 /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 tcggtaccct gtgtgtttgt gcatgtttct ttcatataca 2701 tttgcaaaaa ttgcccacca cagctccatt ttgaaattaa gaaccctgca tcaagttgta 2761 ctgtcagtaa tggggaaagg actttcattg taaaaattca ctttctttct gttaacattt 2821 ttttgtatct gagagtttta cattaatgaa cagcatgtca gaagtacaaa tttggtgaaa 2881 ggaacagagg ctcatctatt tgagtcttgc ccagagtcac acagcttact tatgtaaatg 2941 gcagagcaaa actcaaatgc agggcttctg tctgcaaatc ctgtgccttt tttcattttg 3001 ttttgtttgt ctgtctttat accacactgc tgcttatcgg acctaaggct gcaactttgc 3061 cttcatattt atgatgcttt taaataatat taatatttaa tctttattaa gaacccaatg 3121 tggtaggcac tgttattcct attttctaaa gaaagaaact gaggcccaga gaagtaaaca 3181 gcttgaccaa gtcacacatg tggtaagtag ggcagcccaa gtttgaacca ttcagtctac 3241 ctcagggccc accacgctcc tggccactta actgtgccgc tctaaccagc tgagcgaacc 3301 tgtgtgcaga aggcattgtc tgtctggcct cagagtattt gtttttcata tagtcataac 3361 tatgctcaaa ttttgtaatt taaaatatgg gtctttagga cataaagtga aagaacgtgt 3421 gtgatttggc tcaagatata gattgctttc tataccataa aatctgtagc tcagattcct 3481 ttaaaaaatg aaactctctc atactttcag aaagagggaa ggcctcataa gcattttcca 3541 aattagaatt tttaaaccag agagtgaaag tgatatgcat tatgtagaat tttaaggatt 3601 ttgtccctat ggacagggga gagtgtgagt aactattatg tgggtgccat aaagggagta 3661 aagcaatgct gcataatcca tttgtttttt agacttctag gttgctccat gggaatccct 3721 caactcaact cattctctct gctgcatttc ctctacaaca gagcaccttc cctcagtcac 3781 atcaccagca acatcagtct cagcaacagc agcaactcag ccggcacagg actgacagct 3841 tgcccgaccc ttccaaggtt caaccacagg ggattccaac tactgcaagc gagaatcttt 3901 attttcaggg cgccgccaaa ttcaaagaaa ccgctgctgc taaattcgaa cgccagcaca 3961 tggacagcgg aggtggaggt tcgagcggtc cctcgggttc gtcgagcctg gaagttctgt 4021 tccaggggcc cctgtcgtcg agcggtccct cgggttcgat ggtgagcaaa ggcgaggagc 4081 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 4141 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 4201 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 4261 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 4321 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 4381 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 4441 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 4501 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 4561 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 4621 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 4681 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct taaggagttc gtgaccgccg 4741 ccgggatcac tctcggcatg gacgagctgt acaagtaatg atctagagtc gagttaatta 4801 agaattccgc ccccccccct ctccctcccc ccccctaacg ttactggccg aagccgcttg 4861 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc 4921 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc 4981 cctctcgcca aaggaatgca gggtctgttg aatgtcgtga aggaagcagt tcctctggaa 5041 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct 5101 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca 5161 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca 5221 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat 5281 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc 5341 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac 5401 ctgttacatt gcacaagata aaaatatatc atcacgaaca gtaaaactgt ctgcttacat 5461 aaacagtaat acaaggggtg ttatgggatc ggccattgaa caagatggat tgcacgcagg 5521 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 5581 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 5641 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 5701 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 5761 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 5821 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 5881 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 5941 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 6001 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 6061 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 6121 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 6181 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 6241 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagtcc acacgtgctt 6301 cctctcttga catcaaggga ggaaggggat ggcccattaa gagttactca gatgacctga 6361 ggaaaggagg gaaagttcca gcagtttcat gagatgcagt attgagtgtt ctagttcctg 6421 gaattagttg gcagagaaaa tgctgcctag tgctacagat gtacattaaa taccagccag 6481 caggaggtga tcataggggc atagccagtt ctgacagtgt tttaggtgcc tggatatttt 6541 ttgatggaaa aagaatatat tgccaaatat taagaagctc agctatgaaa tgacctccag 6601 ggaatcagaa aggcactaat gatgttagta acttttagtg gttctgtgcc tcttatcaag 6661 tgttacagag gacataccac tgccatgtca ggggtttgct tacagtgatg ccatgaagac 6721 agtccagtag acttggtagc gaccccctcc cccaacccct ctcccttttc agataatgat 6781 ggaacagtaa ttactttcag aatgttgtgt gggttcaaat tctctatgta cagatgatgt 6841 aaaaatatgt atatgtctag ataaaaggag agaaagcaaa acattttgta tgctgcatga 6901 aagcgttatc tcttccttac aggtgtgagc acctttcctg aaattctgac accatgtgca 6961 aactgatcca tcctgttttt ccttttgttt acaacacagt agtgttctgt tcacttttcc 7021 ggggcacaag tttttttgtt catactttgg ctgtgatgtc acagtttgtt cagtgaggta 7081 tgatgtgctg ctgggaatgg att //