LOCUS Exported 7128 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of CLIP-tagged Neurokinin-1 Receptor in Mammalian cells. ACCESSION . VERSION . KEYWORDS pCLIPf-NK1R Control Plasmid SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7128) AUTHORS Ana Egana TITLE Egana Lab NEB plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 7128) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7128 /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 999..1544 /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="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA VKEWLLAHEGHRLGKPGLG" CDS 2730..2753 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2784..2813 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" intron 2904..3133 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 3116..3135 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 3212..3779 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(3373..3390) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3600..3619 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3815..4618 /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(3878..3897) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4488..4507 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4748..4869 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4785..4804) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4971..4987) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4971..4987) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4984..5006) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4995..5011 /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(5019..5049) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5064..5085 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5202..5219) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5373..5961) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5453..5472) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6132..6992) /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 6755..6774 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6993..7097) /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 cgatatcacc atgcggctct gcatcccgca ggtgctgttg gccttgttcc 961 tttccatgct gacagggccg ggagaaggca gcgctagcat ggacaaagac tgcgaaatga 1021 agcgcaccac cctggatagc cctctgggca agctggaact gtctgggtgc gaacagggcc 1081 tgcaccgtat catcttcctg ggcaaaggaa catctgccgc cgacgccgtg gaagtgcctg 1141 ccccagccgc cgtgctgggc ggaccagagc cactgatcca ggccaccgcc tggctcaacg 1201 cctactttca ccagcctgag gccatcgagg agttccctgt gccagccctg caccacccag 1261 tgttccagca ggagagcttt acccgccagg tgctgtggaa actgctgaaa gtggtgaagt 1321 tcggagaggt catcagcgag agccacctgg ccgccctggt gggcaatccc gccgccaccg 1381 ccgccgtgaa caccgccctg gacggaaatc ccgtgcccat tctgatcccc tgccaccggg 1441 tggtgcaggg cgacagcgac gtggggccct acctgggcgg gctcgccgtg aaagagtggc 1501 tgctggccca cgagggccac agactgggca agcctgggct gggtcctgca ggttccacta 1561 acacctcgga acccaatcag ttcgtgcaac cagcctggca aattgtcctt tgggcagctg 1621 cctacacggt cattgtggtg acctctgtgg tgggcaacgt ggtagtgatg tggatcatct 1681 tagcccacaa aagaatgagg acagtgacga actattttct ggtgaacctg gccttcgcgg 1741 aggcctccat ggctgcattc aatacagtgg tgaacttcac ctatgctgtc cacaacgaat 1801 ggtactacgg cctgttctac tgcaagttcc acaacttctt tcccatcgcc gctgtcttcg 1861 ccagtatcta ctccatgacg gctgtggcct ttgataggta catggccatc atacatcccc 1921 tccagccccg gctgtcagcc acagccacca aagtggtcat ctgtgtcatc tgggtcctgg 1981 ctctcctgct ggccttcccc cagggctact actcaaccac agagaccatg cccagcagag 2041 tcgtgtgcat gatcgaatgg ccagagcatc cgaacaagat ttatgagaaa gtgtaccaca 2101 tctgtgtgac tgtgctgatc tacttcctcc ccctgctggt gattggctat gcatacaccg 2161 tagtgggaat cacactatgg gccagtgaga tccccgggga ctcctctgac cgctaccacg 2221 agcaagtctc tgccaagcgc aaggtggtca aaatgatgat tgtcgtggtg tgcaccttcg 2281 ccatctgcag gctgcccttc cacatcttct tcctcctgcc ctacaccaac ccagatctct 2341 acctgaagaa gtttatccag caggtctacc tggccatcat gtggctggcc atgagctcca 2401 ccatgtacaa ccccatcatc tactgctgcc tcaatgacag gttccgtctg ggcttcaagc 2461 atgccttccg gtgctgcccc ttcatcagcg ccggcgacta tgaggggctg gaaatgaaat 2521 ccacccggta tctccagacc cagggcagtg tgtacaaagt cagccgcctg gagaccacca 2581 tctccacagt ggtgggggcc cacgaggagg agccagagga cggccccaag gccacaccct 2641 cgtccctgga cctgacctcc aactgctctt cacgaagtga ctccaagacc atgacagaga 2701 gcttcagctt ctcctccaat gtgctctccg actacaagga cgacgatgac aagaccggtg 2761 tgccgcgcgg cagcggcagc agccatcatc atcatcatca tcaccatcac cattaaggat 2821 cctgactcga gtgaggcggc cgcatagata actgatccag tgtgctggaa ttaattcgct 2881 gtctgcgagg gccagctgtt ggggtgagta ctccctctca aaagcgggca tgacttctgc 2941 gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat 3001 gcctttgagg gtggccgcgt ccatctggtc agaaaagaca atctttttgt tgtcaagctt 3061 gaggtgtggc aggcttgaga tctggccata cacttgagtg acaatgacat ccactttgcc 3121 tttctctcca caggtgtcca ctcccaggtc caactgcagg tcgagcatgc atctagggcg 3181 gccaattccg cccctctccc cccccccctt ttccctcccc cccccctaac gttactggcc 3241 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc 3301 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta 3361 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag 3421 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga 3481 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg 3541 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 3601 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 3661 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa 3721 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataag 3781 cttgccacaa cccgggataa ttcctgcagc caatatggga tcggccattg aacaagatgg 3841 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 3901 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 3961 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 4021 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 4081 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 4141 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 4201 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 4261 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 4321 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt 4381 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 4441 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 4501 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 4561 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg 4621 ggatcaattc tctagataac tgatcataat cagccatacc acatttgtag aggttttact 4681 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt 4741 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 4801 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 4861 tgtatcttaa cgcgtcgagt gcattctagt tgtggtttgt ccaaactcat caatgtatct 4921 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 4981 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 5041 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 5101 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5161 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5221 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5281 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5341 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5401 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5461 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5521 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 5581 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5641 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5701 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5761 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 5821 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 5881 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 5941 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 6001 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 6061 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 6121 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 6181 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 6241 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 6301 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 6361 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 6421 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 6481 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 6541 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 6601 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 6661 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 6721 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 6781 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 6841 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 6901 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 6961 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 7021 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 7081 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc //