LOCUS Exported 7194 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor SPDEF with GFP. ACCESSION . VERSION . KEYWORDS pSPDEF-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7194) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7194) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7194 /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 3233..3244 /codon_start=1 /product="WELQut protease recognition and cleavage site" /label=WELQut site /translation="WELQ" CDS 3874..3894 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3907..3951 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3991..4014 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 4042..4761 /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(4087..4108) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4348..4367) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4799..5384 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4965..4982) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5192..5211 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5466..6269 /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(5529..5548) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6139..6158 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 gacctgcagg catgcaagct aggcgtaatc 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 tgccaatgaa gccgggactg gcctcccatt 2701 cccactcacc gccaccctcc ctggccaccc agcctcgacc agtgaggaga gctggaccga 2761 cagcgaggtg gactcatcat gctccgggca gcccatccac ctgtggcagt tcctcaagga 2821 gttgctactc aagccccaca gctatggccg cttcattagg tggctcaaca aggagaaggg 2881 tgagcagggt gcccaagggg cgctgcaggg ccagtgagca tggaggttgt gggggcttct 2941 gccaggcacg gtgcatgggg gcaggggcgg gcagaggggt gaagctgatc cccacctttg 3001 tcctagatgc caaatatgca gctctcatgg gacagccctg gggtctgtgt gcaatctgtg 3061 tccacatctg tctcctgcta gactctgtct cacttaggag ttttagtact gcacagcacc 3121 tggaacgagc tccaaatcca ctctccctcc accatctcat attaattaat cctgccagag 3181 agggctgcac aggccgaact gcctgagaat agcaaagagg ttgtttcagg cttgggaact 3241 acagagacac ctgtaatggg gaagggatgc tcttgccaag tgagccgtgg gcactgctgg 3301 gagccacaca ggactttgca ttaggtcatg caaaccccac acagaagcag gagttggcaa 3361 gggccaggcc tgcagggccc cagggaaccc actaaggtac acaggctcac ctgtaccggg 3421 aggtttgtga ctccagagta ttctctgcca ttcactgggg cctgtggcac ctgctttgca 3481 cacgtctctt taacctcacc gcaactacac cttgggagta ggcatttggg cccccacacc 3541 ccgccccacc cgagcagggg gaggcctctg cacggggtcc cttggttcgc cacgaggctg 3601 acctggtggt ctgcgtcttt ctccttcttg ccacctcact cataccactg gctctctcgg 3661 ggccctcctg ctctaggcat cttcaaaatt gaggactcag cccaggtggc ccggctgtgg 3721 ggcatccgca agaaccgtcc cgccatgaac tacgacaagc tgagccgctc catccgccag 3781 tattacaaga agggcatcat ccggaagcca gacatctccc agcgcctcgt ctaccagttc 3841 gtgcacccca tcgggattcc aactactgca agcgagaatc tttattttca gggcgccgcc 3901 aaattcaaag aaaccgctgc tgctaaattc gaacgccagc acatggacag cggaggtgga 3961 ggttcgagcg gtccctcggg ttcgtcgagc ctggaagttc tgttccaggg gcccctgtcg 4021 tcgagcggtc cctcgggttc gatggtgagc aaaggcgagg agctgttcac cggggtggtg 4081 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 4141 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 4201 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 4261 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 4321 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 4381 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 4441 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 4501 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 4561 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 4621 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 4681 gagaagcgcg atcacatggt ccttaaggag ttcgtgaccg ccgccgggat cactctcggc 4741 atggacgagc tgtacaagta atgatctaga gtcgagttaa ttaagaattc cgcccccccc 4801 cctctccctc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc 4861 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa 4921 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat 4981 gcagggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac 5041 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg 5101 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt 5161 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg 5221 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac 5281 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga 5341 cgtggttttc ctttgaaaaa cacgatgata atatggccac aacctgttac attgcacaag 5401 ataaaaatat atcatcacga acagtaaaac tgtctgctta cataaacagt aatacaaggg 5461 gtgttatggg atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 5521 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 5581 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 5641 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 5701 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 5761 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 5821 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 5881 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 5941 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 6001 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 6061 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 6121 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 6181 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 6241 tctatcgcct tcttgacgag ttcttctgag tcgtgcctgg cccagggcct gaaacccgcc 6301 ctcaggggcc tctctcctgc ctgccctgcc tcagccaggc cctgagatgg gggaaaacgg 6361 gcagtctgct ctgctgctct gaccttccag agcccaaggt cagggagggg caaccaactg 6421 ccccaggggg atatgggtcc tctggggcct tcgggaccct ggggcagggg tgcttcctcc 6481 tcaggcccag ctgctcccct ggaggacaga gggagacagg gctgctcccc aacacctgcc 6541 tctgacccca gcatttccag agcagagcct acagaagggc agtgactcga caaaggccac 6601 aggcagtcca ggcctctctc tgctccatcc ccctgcctcc cattctgcac cacacctggc 6661 atggtgcagg gagacatctg cacccctgag ttgggcagcc aggagtgccc ccgggaatgg 6721 ataataaaga tactagagaa ctgacatgtc cccagtgtct gcgcagtgaa tggcacagcc 6781 ccgaggtcat caatggtcaa atgcggctgt ggtcacaggg cctggagtag tcaagggagg 6841 ttttctggag gaggtggctt tgggctgagc ctaaagggca agggataatt gattggtaga 6901 tgcttctggg agagagatag tgaggcttca aaatcaagga ggggctagaa cagccacctc 6961 atcttccctg tcgctgttcg ggctgagagg caggcggcaa ggccggtgct aaagaggtgc 7021 tacatgcagg accggagtgg ggaaggcatc tttcgcatcc ttttgctctg ttgcagagca 7081 gggctttgca gctaacaggg tggaagagag gttggttggc tcctgtttat gtttcctgaa 7141 ggcctcagca gtgctgtctg tgaagatgtg agctcctgtg agtgggtgtg agac //