LOCUS Exported 7152 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor ISL1 with GFP. ACCESSION . VERSION . KEYWORDS pISL1-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7152) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 7152) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7152 /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 3761..3781 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3794..3838 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3878..3901 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 3929..4648 /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(3974..3995) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4235..4254) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4686..5271 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4852..4869) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5079..5098 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5353..6156 /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(5416..5435) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6026..6045 /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 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 tcggtaccca gtgggtgcag aggctagaag tcccttaatc 2701 tcatattgga aaaatttact gtagaaagaa atgtaggctc tagaactagg aaaaaaaaat 2761 tattctaagc tcattaatct gttgagttat ttgagcgaat cctgaatcac aggaggaagg 2821 taaggggagg gttcagggca gccaaatgtt tgcactttct gaaactttag tgtcagatga 2881 gagcagtgga agggaagctg aggcaggagt gggcatagtt agagaaggtt tacaacagca 2941 gtacaatgcg tttagggtta aaagaaggag tcagatattt aagaaggagt cagatattag 3001 ggttaaaaga aggagtcaga atgggatgat gtcataatat atgggtctca ttttggaagg 3061 aagagcctga tttaaagaga gagagagaga aaggccaaaa ggcagcagga ccaacaagga 3121 agaatgccca agctgtgagc ctgctgagga gttaatcttt gttctgtgga gcctcctctc 3181 aatctcctgt caaaggatct gagcctgtta cggattttcc aactgaagaa gagagtcttt 3241 gatgcctaga gactgagagc tcacctactc ccagggcaac atgtagccag caggataatt 3301 ttatttcgag catgcatagt agagttgtga tgccatttta cagtgggaaa cacatttgtt 3361 cttaaataat ttaatgcaac ataatgttgg gaattcagtt tcagttaaaa cagagatctt 3421 ttggaagatg ggaaagtgag aggatttctt cccaagtttt tctcctctag gctttctcta 3481 agcctgttaa aattcagtta tctatgtgaa tatctttaca tatctatcta cacaaacatt 3541 tctacatata caatatgatg agtttataat ctttttatga atactattcc agtgtccttt 3601 atttatttct caaccttcta tgcaggtcaa tttttcagaa ggaggaccgg gctctaattc 3661 cactggcagt gaagtagcat caatgtcctc tcaacttcca gatacaccta acagcatggt 3721 agccagtcct attgaggcag ggattccaac tactgcaagc gagaatcttt attttcaggg 3781 cgccgccaaa ttcaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagcgg 3841 aggtggaggt tcgagcggtc cctcgggttc gtcgagcctg gaagttctgt tccaggggcc 3901 cctgtcgtcg agcggtccct cgggttcgat ggtgagcaaa ggcgaggagc tgttcaccgg 3961 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 4021 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 4081 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 4141 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 4201 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 4261 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 4321 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 4381 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 4441 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 4501 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 4561 ccccaacgag aagcgcgatc acatggtcct taaggagttc gtgaccgccg ccgggatcac 4621 tctcggcatg gacgagctgt acaagtaatg atctagagtc gagttaatta agaattccgc 4681 ccccccccct ctccctcccc ccccctaacg ttactggccg aagccgcttg gaataaggcc 4741 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 4801 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 4861 aaggaatgca gggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 4921 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 4981 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 5041 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 5101 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 5161 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc 5221 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac ctgttacatt 5281 gcacaagata aaaatatatc atcacgaaca gtaaaactgt ctgcttacat aaacagtaat 5341 acaaggggtg ttatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc 5401 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 5461 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 5521 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 5581 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 5641 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 5701 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 5761 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 5821 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 5881 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 5941 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 6001 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 6061 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 6121 atcgccttct atcgccttct tgacgagttc ttctgagtcg gaacattcat tctgtatttt 6181 ttttccctgt tggagaaagt gggaaattat aatgtcgaac tctgaaacaa aagtatttaa 6241 cgacccagtc aatgaaaact gaatcaagaa atgaatgctc catgaaatgc acgaagtctg 6301 ttttaatgac aaggtgatat ggtagcaaca ctgtgaagac aatcatggga ttttactaga 6361 attaaacaac aaacaaaacg caaaacccag tatatgctat tcaatgatct tagaagtact 6421 gaaaaaaaaa gacgttttta aaacgtagag gatttatatt caaggatctc aaagaaagca 6481 ttttcatttc actgcacatc tagagaaaaa caaaaataga aaattttcta gtccatccta 6541 atctgaatgg tgctgtttct atattggtca ttgccttgcc aaacaggagc tccagcaaaa 6601 gcgcaggaag agagactggc ctccttggct gaaagagtcc tttcaggaag gtggagctgc 6661 attggtttga tatgtttaaa gttgacttta acaaggggtt aattgaaatc ctgggtctct 6721 tggcctgtcc tgtagctggt ttatttttta ctttgccccc tccccacttt ttttgagatc 6781 catcctttat caagaagtct gaagcgactt taaaggtttt tgaattcaga tttaaaaacc 6841 aacttataaa gcattgcaac aaggttacct ctattttgcc acaagcgtct cgggattgtg 6901 tttgacttgt gtctgtccaa gaacttttcc cccaaagatg tgtatagtta ttggttaaaa 6961 tgactgtttt ctctctctat ggaaataaaa aggaaaaaaa aaaaggaaac tttttttgtt 7021 tgctcttgca ttgcaaaaat tataaagtaa tttattattt attgtcggaa gacttgccac 7081 ttttcatgtc atttgacatt ttttgtttgc tgaagtgaaa aaaaaagata aaggttgtac 7141 ggtggtcttt ga //