LOCUS Exported 5724 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pMLM290 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5724) TITLE OPEN plasmids REFERENCE 2 (bases 1 to 5724) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5724 /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 MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR YVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVL 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..2727 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2578..2713 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2640..2659 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 2775..2822 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 2841..3239 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 3397..3518 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3434..3453) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3488..3507 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3567..3583) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3567..3583) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3580..3602) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3591..3607 /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(3615..3645) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3660..3681 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3798..3815) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3969..4557) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4049..4068) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4728..5588) /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 5351..5370 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5589..5693) /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 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 gcaacgatat gtcaaagaaa atcaaacacg 1321 aaacaaacat atcaacccta atgaatggtg gaaagtctat ccatcttctg taacggaatt 1381 taagttttta tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt 1441 aaatcataag 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 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 2161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 2221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 2281 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 2341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 2401 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2461 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt 2521 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 2581 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 2641 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc 2701 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga 2761 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg 2821 acaaggtgag gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg 2881 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg 2941 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg 3001 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga 3061 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc 3121 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac 3181 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag 3241 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc 3301 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 3361 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 3421 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 3481 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 3541 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 3601 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 3661 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 3721 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 3781 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 3841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 3901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 3961 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 4021 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 4081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 4141 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 4201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 4261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 4321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 4381 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 4441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 4501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 4561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 4621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 4681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 4741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 4801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 4861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 4921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 4981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 5041 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 5101 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 5161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 5221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 5281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 5341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 5401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 5461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 5521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 5581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 5641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 5701 ccccgaaaag tgccacctga cgtc //