LOCUS Exported 5805 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of protein of interest with CLIP-Tag in Mammalian cells . ACCESSION . VERSION . KEYWORDS pCLIP-tag (m) Vector SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5805) AUTHORS Ana Egana TITLE Egana Lab NEB plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5805) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5805 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 930..1475 /codon_start=1 /product="human O6-alkylguanine-DNA-alkyltransferase variant that forms covalent bonds with benzylcytosine derivatives" /label=CLIP-tag(TM) /note="optimized for mammalian expression" /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIIFLGKGTSAADAVE VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA VKEWLLAHEGHRLGKPGLG" intron 1581..1810 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 1793..1812 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 1889..2456 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2050..2067) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2277..2296 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2492..3295 /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 EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2555..2574) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3165..3184 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3425..3546 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3462..3481) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(3648..3664) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3648..3664) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3661..3683) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3672..3688 /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(3696..3726) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3741..3762 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3879..3896) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4050..4638) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4130..4149) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4809..5669) /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 5432..5451 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5670..5774) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 901 gagctcggat cgatatcgaa ttcgctagca tggacaaaga ctgcgaaatg aagcgcacca 961 ccctggatag ccctctgggc aagctggaac tgtctgggtg cgaacagggc ctgcacgaga 1021 tcatcttcct gggcaaagga acatctgccg ccgacgccgt ggaagtgcct gccccagccg 1081 ccgtgctggg cggaccagag ccactgatcc aggccaccgc ctggctcaac gcctactttc 1141 accagcctga ggccatcgag gagttccctg tgccagccct gcaccaccca gtgttccagc 1201 aggagagctt tacccgccag gtgctgtgga aactgctgaa agtggtgaag ttcggagagg 1261 tcatcagcga gagccacctg gccgccctgg tgggcaatcc cgccgccacc gccgccgtga 1321 acaccgccct ggacggaaat cccgtgccca ttctgatccc ctgccaccgg gtggtgcagg 1381 gcgacagcga cgtggggccc tacctgggcg ggctcgccgt gaaagagtgg ctgctggccc 1441 acgagggcca cagactgggc aagcctgggc tgggtcctgc aggtataggc gcgccaggat 1501 ccctcgagtg aggcggccgc atagataact gatccagtgt gctggaatta attcgctgtc 1561 tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1621 aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg cggtgatgcc 1681 tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt caagcttgag 1741 gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca ctttgccttt 1801 ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc tagggcggcc 1861 aattccgccc ctctcccccc cccccttttc cctccccccc ccctaacgtt actggccgaa 1921 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt 1981 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg 2041 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc 2101 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc 2161 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa 2221 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc 2281 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg 2341 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac 2401 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt 2461 gccacaaccc gggataattc ctgcagccaa tatgggatcg gccattgaac aagatggatt 2521 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 2581 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 2641 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 2701 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 2761 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 2821 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 2881 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 2941 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 3001 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgatgatc tcgtcgtgac 3061 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 3121 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 3181 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 3241 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagggga 3301 tcaattctct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc 3361 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 3421 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 3481 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 3541 atcttaacgc gtcgagtgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 3601 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt 3661 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 3721 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 3781 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 3841 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 3901 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 3961 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4021 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4081 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4141 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4201 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4261 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4321 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4381 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4441 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4501 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4561 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4621 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4681 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 4741 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 4801 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 4861 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 4921 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 4981 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5041 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5101 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5161 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5221 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5281 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5341 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5401 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5461 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 5521 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5581 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 5641 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 5701 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 5761 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc //