LOCUS Exported 5802 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of protein of interest with SNAP-Tag in Mammalian cells . ACCESSION . VERSION . KEYWORDS pSNAP-tag (m) Vector SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5802) AUTHORS Ana Egana TITLE Egana Lab NEB plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5802) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5802 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(159..176) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 386..405 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 601..1404 /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(664..683) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1274..1293 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 1534..1655 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1571..1590) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(1757..1773) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1757..1773) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1770..1792) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1781..1797 /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(1805..1835) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1850..1871 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1988..2005) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2159..2747) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2239..2258) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2918..3778) /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 3541..3560 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3779..3883) /gene="bla" /label=AmpR promoter primer_bind complement(3958..3977) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 4149..4528 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4529..4732 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 4683..4703 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 4777..4796 /label=T7 /note="T7 promoter, forward primer" promoter 4777..4795 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 4841..5386 /codon_start=1 /product="human O6-alkylguanine-DNA-alkyltransferase variant that forms covalent bonds with benzylguanine derivatives" /label=SNAP-tag(R) /note="optimized for mammalian expression" /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA VKEWLLAHEGHRLGKPGLG" intron 5492..5721 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 5704..5723 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 5800..565 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" ORIGIN 1 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 61 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 121 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 181 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 241 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 301 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 361 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 421 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 481 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 541 tttcctttga aaaacacgat gataagcttg ccacaacccg ggataattcc tgcagccaat 601 atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 661 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 721 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 781 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 841 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 901 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 961 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 1021 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 1081 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 1141 atgcccgacg gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 1201 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 1261 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 1321 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 1381 cgccttcttg acgagttctt ctgaggggat caattctcta gataactgat cataatcagc 1441 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 1501 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 1561 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 1621 agttgtggtt tgtccaaact catcaatgta tcttaacgcg tcgagtgcat tctagttgtg 1681 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 1741 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 1801 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 1861 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 1921 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 1981 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 2041 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 2101 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 2161 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 2221 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 2281 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 2341 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 2401 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 2461 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 2521 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 2581 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 2641 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 2701 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 2761 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 2821 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 2881 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 2941 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 3001 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 3061 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 3121 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 3181 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 3241 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 3301 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 3361 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 3421 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 3481 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 3541 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 3601 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3661 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3721 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 3781 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 3841 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 3901 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac 3961 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt 4021 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg 4081 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata 4141 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 4201 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 4261 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 4321 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 4381 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 4441 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 4501 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 4561 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 4621 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 4681 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct 4741 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac 4801 ccaagcttgg taccgagctc ggatcgatat cgaattcacc atggacaaag actgcgaaat 4861 gaagcgcacc accctggata gccctctggg caagctggaa ctgtctgggt gcgaacaggg 4921 cctgcacgag atcaagctgc tgggcaaagg aacatctgcc gccgacgccg tggaagtgcc 4981 tgccccagcc gccgtgctgg gcggaccaga gccactgatg caggccaccg cctggctcaa 5041 cgcctacttt caccagcctg aggccatcga ggagttccct gtgccagccc tgcaccaccc 5101 agtgttccag caggagagct ttacccgcca ggtgctgtgg aaactgctga aagtggtgaa 5161 gttcggagag gtcatcagct accagcagct ggccgccctg gccggcaatc ccgccgccac 5221 cgccgccgtg aaaaccgccc tgagcggaaa tcccgtgccc attctgatcc cctgccaccg 5281 ggtggtgtct agctctggcg ccgtgggggg ctacgagggc gggctcgccg tgaaagagtg 5341 gctgctggcc cacgagggcc acagactggg caagcctggg ctgggtcctg caggtatagg 5401 cgcgccagga tccctcgagt gaggcggccg catagataac tgatccagtg tgctggaatt 5461 aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg 5521 acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc 5581 gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg 5641 tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc 5701 actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc gagcatgcat 5761 ctagggcggc caattccgcc cctctccccc cccccctttt cc //