LOCUS Exported 5855 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION FX cloning mammalian cell line expression vector with CMV promoter and C-terminal 3C protease cleavage site, Myc tag and streptavidin binding peptide. ACCESSION . VERSION . KEYWORDS pcDXC3MS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5855) AUTHORS Geertsma ER, Dutzler R TITLE A versatile and efficient high-throughput cloning tool for structural biology. JOURNAL Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25. PUBMED 21410291 REFERENCE 2 (bases 1 to 5855) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5855 /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" misc_feature 917..1079 /label=QBI SP163 /note="strong translational enhancer derived from the 5' UTR of the mouse VEGF gene" CDS complement(1131..1436) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(1233..1252) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1778..2437) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2352..2371 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2491..2521) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(2546..2670) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(2689..2709) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(2713..2733) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2760..2783 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 2793..2822 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2835..2948 /codon_start=1 /product="streptavidin-binding peptide" /label=SBP /note="selected from a peptide library; binds streptavidin with nanomolar affinity (Keefe et al., 2001)" /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP" primer_bind complement(2983..3000) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2989..3213 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3259..3687 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3346..3365) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3556..3577 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3696..3716) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3701..4030 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3881..4016 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3943..3962 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" rep_origin complement(4103..4688) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4183..4202) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4859..5719) /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 5482..5501 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5720..5824) /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 gctggctagc 901 gtttaaactt aagcttagcg cagaggcttg gggcagccga gcggcagcca ggccccggcc 961 cgggcctcgg ttccagaagg gagaggagcc cgccaaggcg cgcaagagag cgggctgcct 1021 cgcagtccga gccggagagg gagcgcgagc cgcgccggcc ccggacggcc tccgaaacca 1081 tgagtagaag agcgagctgc agactggctg tgtataaggg agcctgacat ttatattccc 1141 cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact 1201 tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat gcgccagctt 1261 tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt 1321 gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca 1381 tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatttca 1441 ccagcccctg ttctcgtcag caaaagagcc gttcatttca ataaaccggg cgacctcagc 1501 catcccttcc tgattttccg ctttccagcg ttcggcacgc agacgacggg cttcattctg 1561 catggttgtg cttaccagac cggagatatt gacatcatat atgccttgag caactgatag 1621 ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag catacctctt tttgacatac 1681 ttcgggtata catatcagta tatattctta taccgcaaaa atcagcgcgc aaatacgcat 1741 actgttatct ggcttttagt aagccggatc cacgcgttta cgccccgccc tgccactcat 1801 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat 1861 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 1921 tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga 1981 aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat 2041 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga 2101 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg 2161 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga 2221 attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt 2281 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat 2341 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata 2401 tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa 2461 atctcgacgg atcctaactc aaaatccaca cattatacga gccggaagca taaagtgtaa 2521 agcctggggt gcctaatgcg gccgccatag tgactggata tgttgtgttt tacagtatta 2581 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt 2641 ttctcgttca gcttttttgt acaaacttgt gatatcgaat tcgctagcta cctttctctt 2701 cttttttgga tctacctttc tcttcttttt tggggatcca ctagttcagc tcttctgcat 2761 tagaagtttt gtttcaaggt ccacaaggta ccgaacaaaa actcatctca gaagaggatc 2821 tgaggggggc cagcatggac gagaagacca ctggttggcg aggtggacac gttgttgaag 2881 gactggctgg ggaacttgaa caacttcgtg cacgactgga gcatcaccca caaggtcaac 2941 gtgaaccata atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 3001 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 3061 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 3121 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 3181 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 3241 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 3301 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 3361 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 3421 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 3481 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 3541 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 3601 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 3661 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg 3721 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 3781 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3841 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3901 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3961 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 4021 cttttgcaaa aagctcccgg gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4621 aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 4681 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 4741 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 4801 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 4861 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 4921 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 4981 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 5041 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 5101 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 5161 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 5221 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 5281 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 5341 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 5401 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 5461 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 5521 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 5581 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 5641 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 5701 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 5761 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 5821 cgcgcacatt tccccgaaaa gtgccacctg acgtc //