LOCUS Exported 5725 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pMLM292 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5725) TITLE OPEN plasmids REFERENCE 2 (bases 1 to 5725) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5725 /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 910..930 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 940..963 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 986..1573 /codon_start=1 /product="nonspecific DNA cleavage domain of the FokI endonuclease (Li et al., 1992)" /label=FokI cleavage domain /note="must dimerize to be catalytically active" /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMER YVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" CDS 1589..1630 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 1640..1657 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1680..1697) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1686..1910 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 1956..2384 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2043..2062) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2253..2274 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2393..2413) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2398..2728 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2579..2714 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2641..2660 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 2776..2823 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 2842..3240 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 3398..3519 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3435..3454) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3489..3508 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3568..3584) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3568..3584) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3581..3603) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3592..3608 /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(3616..3646) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3661..3682 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3799..3816) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3970..4558) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4050..4069) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4729..5589) /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 5352..5371 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5590..5694) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 accatgggac ctaagaaaaa gaggaaggtg gcggccgctg actacaagga tgacgacgat 961 aaatctagat agtaagcttg gatcccaact agtcaaaagt gaactggagg agaagaaatc 1021 tgaacttcgt cataaattga aatatgtgcc tcatgaatat attgaattaa ttgaaattgc 1081 cagaaattcc actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt 1141 ttatggatat agaggtaaac atttgggtgg atcaaggaaa ccggacggag caatttatac 1201 tgtcggatct cctattgatt acggtgtgat cgtggatact aaagcttata gcggaggtta 1261 taatctgcca attggccaag cagatgaaat ggagcgatat gtcgaagaaa atcaaacacg 1321 aaacaaacat ctcaacccta atgaatggtg gaaagtctat ccatcttctg taacggaatt 1381 taagttttta tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt 1441 aaatcatatc actaattgta atggagctgt tcttagtgta gaagagcttt taattggtgg 1501 agaaatgatt aaagccggca cattaacctt agaggaagtg agacggaaat ttaataacgg 1561 cgagataaac ttttaagggc ccttcgaagg taagcctatc cctaaccctc tcctcggtct 1621 cgattctacg cgtaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc 1681 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 1741 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 1801 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 1861 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 1921 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 1981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 2041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 2101 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 2161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 2221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 2281 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta 2341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 2401 tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat 2461 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 2521 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 2581 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 2641 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 2701 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 2761 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac 2821 gacaaggtga ggaactaaac catggccaag cctttgtctc aagaagaatc caccctcatt 2881 gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc 2941 gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg 3001 ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg 3061 acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgt 3121 cgacaggtgc ttctcgatct gcatcctggg atcaaagcga tagtgaagga cagtgatgga 3181 cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa 3241 gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc 3301 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 3361 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 3421 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 3481 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 3541 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 3601 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 3661 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3721 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3781 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 3841 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 3901 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 3961 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4021 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4081 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4141 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 4201 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4261 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4321 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4381 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 4441 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4501 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4561 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4621 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 4681 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 4741 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 4801 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 4861 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 4921 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 4981 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5041 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5101 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5161 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 5221 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 5281 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5341 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5401 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5461 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5521 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 5581 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 5641 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 5701 tccccgaaaa gtgccacctg acgtc //