LOCUS Exported 6300 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Destination vector for TALE-KRAB construction. ACCESSION . VERSION . KEYWORDS pTALE-STAR-KRAB-T SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6300) AUTHORS Gogolok S, Garcia-Diaz C, Pollard SM TITLE STAR: a simple TAL effector assembly reaction using isothermal assembly. JOURNAL Sci Rep. 2016 Sep 12;6:33209. doi: 10.1038/srep33209. PUBMED 27615025 REFERENCE 2 (bases 1 to 6300) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6300 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 383..658 /label=chicken beta-actin promoter intron 659..1667 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1591..1613 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" regulatory 1731..1740 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1740..1805 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1812..1832 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2615..2635 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2690..2884 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEPWLV" primer_bind complement(2907..2924) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2913..3137 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3184..3200) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3184..3200) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3197..3219) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3208..3224 /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(3232..3262) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3277..3298 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3357..3552 /label=SV40 promoter /note="SV40 early promoter" rep_origin 3403..3538 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3465..3484 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 3558..3692 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3760..3777) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3931..4519) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4011..4030) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4690..5550) /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 5313..5332 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" polyA_signal 6153..6287 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc 421 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 481 atgggggcgg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 541 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 601 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 661 agtcgctgcg ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg 721 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg 781 ctgtaattag cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct 841 taaagggctc cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt 901 gtgtgtgcgt ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg 961 gcgcggcgcg gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt 1021 gccccgcggt gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1081 gggggtgagc agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct 1141 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg 1201 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 1261 cctcgggccg gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc 1321 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac 1381 ttcctttgtc ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag 1441 cgggcgcggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt 1501 gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg 1561 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct 1621 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg 1681 tgctggttgt tgtgctgtct catcattttg gcaaagaatt cctcgaggcc gccaccatgg 1741 actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg 1801 acaagatggc ccccaagaag aagaggaagg tgggcattca ccgcggggta cctatggtgg 1861 acttgaggac actcggttat tcgcaacagc aacaggagaa aatcaagcct aaggtcagga 1921 gcaccgtcgc gcaacaccac gaggcgcttg tggggcatgg cttcactcat gcgcatattg 1981 tcgcgctttc acagcaccct gcggcgcttg ggacggtggc tgtcaaatac caagatatga 2041 ttgcggccct gcccgaagcc acgcacgagg caattgtagg ggtcggtaaa cagtggtcgg 2101 gagcgcgagc acttgaggcg ctgctgactg tggcgggtga gcttaggggg cctccgctcc 2161 agctcgacac cgggcagctg ctgaagatcg cgaagagagg gggagtaaca gcggtagagg 2221 cagtgcacgc ctggcgcaat gcgctcaccg gggccccctt gaacctgcag gatacgttta 2281 tcagtgataa agtgtcaagc atgacaaagt tgcagccgaa tacagtggta tcctgcaggt 2341 gctctgtcag gaccacgggc ttacgccaca acaggtggtg gcgattgcat ctaatggagg 2401 cggacgccct gccttggagt ccatcgtagc ccaattgtcc aggcccgatc ccgcgttggc 2461 tgcgttaacg aatgaccatc tggtggcgtt ggcatgtctt ggtggacgac ccgcgctcga 2521 tgcagtcaaa aagggtctgc ctcatgctcc cgcattgatc aaaagaacca accggcggat 2581 tcccgagaga acttcccatc gagtcgcggc tagccccaag aagaagagaa aggtggaggc 2641 cagcggttcc ggacagtgga tggacgcgaa atcacttacg gcatggtcga gaacactggt 2701 tacgttcaag gacgtgtttg tggactttac acgtgaggag tggaaattgc tggatactgc 2761 gcaacaaatt gtgtatcgaa atgtcatgct tgagaattac aagaacctcg tcagtctcgg 2821 ataccagttg acgaaaccgg atgtgatcct taggctcgaa aagggggaag aaccttggct 2881 ggtataaagc tgagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 2941 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 3001 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 3061 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 3121 tgcggtgggc tctatggctt ctgaggcgga aagaacctgc agcccaagct tggcgtaatc 3181 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 3241 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 3301 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat 3361 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 3421 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 3481 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 3541 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata 3601 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 3661 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc 3721 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3781 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3841 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3901 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3961 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 4021 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgc 4081 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 4141 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 4201 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 4261 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 4321 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 4381 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4441 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4501 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4561 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4621 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4681 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4741 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4801 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4861 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 4921 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 4981 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 5041 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 5101 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 5161 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 5221 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 5281 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 5341 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 5401 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 5461 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5521 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatgac cgctttggaa 5581 aaacaaagac tgtatttcct ggaaattaat gtttattcaa taaactgtgt attcagctat 5641 atcacatagt ggtgaggctg aaatgaggcg ggaagaggcg gttggggctt aattatatca 5701 atttgggtgg ccccacagcg cctccaaggc gccagtcctg ttttgacaag ttgcctctgg 5761 aagcctctac aatgcctctc ttctttttct ccagagtaag cggaggccag gggcccccgg 5821 cctctgctta atactaaaaa aaacagctgt tgtcatagta atgattgggt ggaaacattc 5881 caggcctggg tggagaggct ttttgcttcc tcttgcaaaa ccacactgac attccaggcc 5941 tgggtggaga ggctttttgc ttcctcttgc aaaaccacac tgccctctgg agggcagttg 6001 cctagcaact aactaaaaga ggatgtcgca cggccagctg cggtcagtta gtcacttcct 6061 gcttaactga cttgacattt tctattttaa gagtcgggag gaaaattact gtgttggagg 6121 ccctccgcca tcttctgaag ctgaatcgaa ttaacttgtt tattgcagct tataatggtt 6181 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 6241 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga tatcatcgtc //