LOCUS Exported 6773 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR donor plasmid to tag human transcription factor ZNF444 with GFP. ACCESSION . VERSION . KEYWORDS pZNF444-donor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6773) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 6773) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6773 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(19..36) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(19..35) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(28..50) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(244..263) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 363..385 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(423..441) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 509..613 /gene="bla" /label=AmpR promoter CDS 614..1474 /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 complement(832..851) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1645..2233 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2134..2153 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2387..2404 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2521..2542 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2557..2587 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2595..2611 /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)." primer_bind 2600..2622 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2619..2635 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2619..2635 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 3523..3543 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3556..3600 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 3640..3663 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 3691..4410 /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(3736..3757) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3997..4016) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4448..5033 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4614..4631) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4841..4860 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5115..5918 /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(5178..5197) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5788..5807 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 61 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 121 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 181 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 241 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 301 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 361 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 421 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 481 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 541 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 601 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 661 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 721 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 781 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 841 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 901 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 961 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 1021 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 1081 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 1141 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 1201 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 1261 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 1321 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 1381 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 1441 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 1501 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 1561 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 1621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 1681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 1741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 1801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 1861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 1921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 1981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 2041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 2101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 2161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 2221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 2281 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 2341 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 2401 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 2461 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 2521 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 2581 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 2641 ttacgccaag cttatcatcc acccacgcat tcttcatcta cccacgcatt catctaccca 2701 tccatcatcc agccagccat catctattat gtattgggca ttgactgtat gccaggcact 2761 actaggtacc gggacacagg tatgaataag aagcccacag cccagccctc ttcccacccc 2821 aatggtttct gcctttaagc gttccctccc tgcctccacc ccagcctgtg ctgtccttgc 2881 cccgccctcg tgccgaccca gatgctgact ctctttcttt tcccagcagg caccgcccct 2941 ggggctgagg ggccggcgcc tggggactcc caggctgtgc gcccctacaa gcaggagccc 3001 agcagccccc cgctggcgcc tggcctgccc gccttcctag cggccccggg caccacgtcc 3061 tgccccgagt gcggcaaaac gtccctgaaa ccagctcacc tgctgcgcca ccggcagagc 3121 cactcgggcg agaagccgca cgcctgccct gagtgcggga aggcctttcg gcgcaaggag 3181 cacctgcggc gccaccgcga cacgcacccc ggcagccccg gcagccccgg gcccgcgctg 3241 cgccctctgc ccgcccgtga gaagccccac gcgtgctgcg agtgtggcaa gaccttctac 3301 tggcgcgagc acctggtgcg ccaccgcaag acgcactcgg gagcgcggcc ctttgcctgc 3361 tgggagtgtg gcaagggctt cgggcgccgc gagcacgtgc tgcgccacca gcgcatccac 3421 ggccgggcag cggccagcgc gcagggggcg gtagctccgg gcccggatgg tggaggcccc 3481 ttcccgccct ggcccttggg tgggattcca actactgcaa gcgagaatct ttattttcag 3541 ggcgccgcca aattcaaaga aaccgctgct gctaaattcg aacgccagca catggacagc 3601 ggaggtggag gttcgagcgg tccctcgggt tcgtcgagcc tggaagttct gttccagggg 3661 cccctgtcgt cgagcggtcc ctcgggttcg atggtgagca aaggcgagga gctgttcacc 3721 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 3781 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 3841 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag 3901 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 3961 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 4021 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 4081 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 4141 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 4201 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 4261 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa 4321 gaccccaacg agaagcgcga tcacatggtc cttaaggagt tcgtgaccgc cgccgggatc 4381 actctcggca tggacgagct gtacaagtaa tgatctagag tcgagttaat taagaattcc 4441 gccccccccc ctctccctcc ccccccctaa cgttactggc cgaagccgct tggaataagg 4501 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag 4561 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 4621 caaaggaatg cagggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 4681 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 4741 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 4801 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 4861 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 4921 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa 4981 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca acctgttaca 5041 ttgcacaaga taaaaatata tcatcacgaa cagtaaaact gtctgcttac ataaacagta 5101 atacaagggg tgttatggga tcggccattg aacaagatgg attgcacgca ggttctccgg 5161 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 5221 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 5281 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 5341 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 5401 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 5461 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 5521 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 5581 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 5641 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 5701 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 5761 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 5821 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 5881 gcatcgcctt ctatcgcctt cttgacgagt tcttctgacc gcctcccggc cagcgccatc 5941 tcccgccctt ggtgctgccc ccgggcggta cctgctctct cccagcgcca cttggcctct 6001 tcctctcctc cttccctccc atcgtcctcc tccacctgcg cctcccttgt ctgaacttcc 6061 caacgccttc ctattccttt ccaactcctt ttcccccaaa tttcactttc cttctcaggt 6121 ctcacctcag cccccccctt ctccctgatt tctcggcctc tctctctgtg tgaaggggcc 6181 tctccctaat gtctcctcct tcccccctct tctctctcct gcggcccagc ctccctctcc 6241 ctcctccatt cctctctccc tgcccttttc ctgcctgaag agcagaggtg aggacctggg 6301 acccctgagg ggcaggccag gaggagctcg ggcgcaggcc aggccccctt ggtgaagcag 6361 aggctgaagg aaaggggtct gggtcttgtc cctaggaatt ctcctccctc aggagattgg 6421 gggttggggg aggcagcggg tgatggctct gaagctgaac ccagggcctg gctgtggtct 6481 tcttggtctt gctgccccct gtgacccaaa ggcatgggat ggacagagat gcctgccccc 6541 gtgaagctgg ttggggaggg cagttcacca gcatccatag agtaataaag tcactgtgtg 6601 tagacccgga gtctgagctc tgtctggagt ctgtccggca cagccaccct cccagggccg 6661 agcgtctggg gtgcccgggg cttctggggt cagctgtctt ggctcccacc acagactctc 6721 ttggcatgag agaggctctg ggagtctggg cacagctgtg ggaggcagtg cag //