LOCUS Exported 8580 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses rTetR-KRAB for gene silencing and the nuclear H2B-mCherry marker in mammalian cells.. ACCESSION . VERSION . KEYWORDS pEx1-pEF-H2B-mCherry-T2A-rTetR-KRAB-Zeo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8580) AUTHORS Bintu L, Yong J, Antebi YE, McCue K, Kazuki Y, Uno N, Oshimura M, Elowitz MB TITLE Dynamics of epigenetic regulation at the single-cell level. JOURNAL Science. 2016 Feb 12;351(6274):720-4. doi: 10.1126/science.aab2956. PUBMED 26912859 REFERENCE 2 (bases 1 to 8580) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8580 /organism="synthetic DNA construct" /mol_type="other DNA" intron 214..1152 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1109..1129 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" promoter 1185..1203 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" CDS 1264..1641 /codon_start=1 /gene="HIST1H2BJ" /product="human histone H2B" /label=H2B /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP GELAKHAVSEGTKAITKYTSAK" CDS 1648..2355 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1793..1811) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2061..2080 /label=mCherry-F /note="mCherry, forward primer" CDS 2359..2412 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 3061..3186 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the rat zinc finger protein Kid-1 (Witzgall et al., 1994)" /label=KRAB-A /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM" primer_bind complement(3279..3298) /label=T7 /note="T7 promoter, forward primer" promoter complement(3280..3298) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 3572..3693 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin complement(3700..4155) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3837..3858) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4049..4068 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4182..4286 /gene="bla" /label=AmpR promoter protein_bind complement(4296..4329) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 4342..4671 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4522..4657 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4584..4603 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 4719..4766 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 4785..5159 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 5289..5410 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5662..5678) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5663..5679) /label=pBluescriptKS /note="For pBluescript vector" CDS complement(5923..6582) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 6497..6516 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(6583..6685) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS complement(6854..7714) /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 7477..7496 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7884..8472 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8373..8392 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 atgcatgagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga 61 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc 121 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt 181 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg 241 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag 301 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc 361 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc 421 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc 481 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat 541 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc 601 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc 661 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt 721 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga 781 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag 841 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca 901 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg 961 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag 1021 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc 1081 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt 1141 cttccatttc aggtgtcgtg agctagcgat tacgccaagc tcgaaattaa ccctcactaa 1201 agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagcccggg cggatccgcc 1261 accatgccag agccagcgaa gtctgctccc gccccgaaaa agggctccaa gaaggcggtg 1321 actaaggcgc agaagaaagg cggcaagaag cgcaagcgca gccgcaagga gagctattcc 1381 atctatgtgt acaaggttct gaagcaggtc caccctgaca ccggcatttc gtccaaggcc 1441 atgggcatca tgaattcgtt tgtgaacgac attttcgagc gcatcgcagg tgaggcttcc 1501 cgcctggcgc attacaacaa gcgctcgacc atcacctcca gggagatcca gacggccgtg 1561 cgcctgctgc tgcctgggga gttggccaag cacgccgtgt ccgagggtac taaggccatc 1621 accaagtaca ccagcgctaa gcctaggatg gtgagcaagg gcgaggagga taacatggcc 1681 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag 1741 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg 1801 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg 1861 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc 1921 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc 1981 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc 2041 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc 2101 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag 2161 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc 2221 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag 2281 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg 2341 gacgagctgt acaagtctga gggcaggggg agcctcctaa cttgcggaga cgtcgaggag 2401 aatcctggcc catctagact ggacaagagc aaagtcataa acggagctct ggaattactc 2461 aatggtgtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag 2521 cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc 2581 gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac 2641 tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg cgacggggct 2701 aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc 2761 gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg 2821 ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc aaaagaggaa 2881 agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc 2941 gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg 3001 gagaaacagc taaagtgcga aagcggcggg ggtagcggca gcggtagcgc atgcctagca 3061 gtgtcagtga catttgaaga tgtggctgtg ctctttactc gggacgagtg gaagaagctg 3121 gatctgtctc agagaagcct gtaccgtgag gtgatgctgg agaattacag caacctggcc 3181 tccatggcag gattcctgtt taccaaacca aaggtgatct ccctgttgca gcaaggagag 3241 gatccctggt aaggtaccag gtaagtgtac ccaattcgcc ctatagtgag tcgtattaca 3301 attcactcga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg agatccaatt 3361 tttaagtgta taatgtgtta aactactgat tctaattgtt tgtgtatttt agattcacag 3421 tcccaaggct catttcaggc ccctcagtcc tcacagtctg ttcatgatca taatcagcca 3481 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct 3541 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta 3601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 3661 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt 3721 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa 3781 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag 3841 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg 3901 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga 3961 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg 4021 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg 4081 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc 4141 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4201 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 4261 ttcaataata ttgaaaaagg aagaatcctg aggccataac ttcgtataat gtatgctata 4321 cgaagttatc ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 4381 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 4441 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 4501 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 4561 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 4621 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 4681 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat 4741 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca 4801 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc 4861 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg 4921 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg 4981 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact 5041 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt 5101 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac 5161 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 5221 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 5281 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 5341 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 5401 atgtatctta tcatgtctga attcgagaat gggcggaact gggcggagtt aggggcggga 5461 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac 5521 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca 5581 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca 5641 cattccacag atcaagctta tcgataccgt cgacctcgac ctccacgggg agagcctgag 5701 caaactggcc tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac 5761 cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg 5821 ggtcgaattt gctttcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta 5881 gcaaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca 5941 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac 6001 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt 6061 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact 6121 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg 6181 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg 6241 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa 6301 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat 6361 acgtaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa 6421 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg 6481 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg 6541 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc 6601 tgaaaatctc gacaactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa 6661 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt 6721 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcactcg 6781 agggggggcc gggccataac ttcgtatatt atccatgggc cccccctcga catgagtaaa 6841 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 6901 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 6961 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 7021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7141 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7201 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7261 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7321 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7381 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7441 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 7501 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 7561 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 7621 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 7681 gaataagggc gacacggaaa tgttgaatac tcatactctt cctcaggtta ctcatatata 7741 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 7801 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 7861 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 7921 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 7981 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 8041 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 8101 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 8161 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 8221 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 8281 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 8341 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 8401 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 8461 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 8521 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc //