LOCUS Exported 7165 bp ds-DNA circular SYN 24-FEB-2020 DEFINITION Parental vector for TOPO(R) cloning of blunt PCR products. ACCESSION . VERSION . KEYWORDS RAD21-mAID-Clover in pCR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7165) AUTHORS Invitrogen (Life Technologies) TITLE Direct Submission JOURNAL Exported Monday, Feb 24, 2020 from SnapGene 5.0.7 https://www.snapgene.com FEATURES Location/Qualifiers source 1..7165 /organism="synthetic DNA construct" /lab_host="Escherichia coli" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." /note="color: #31849b" promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" /note="This forward directional feature has 3 segments: 1: 143 .. 148 / #ffffff / -35 2: 149 .. 166 / #ffffff 3: 167 .. 173 / #ffffff / -10" protein_bind 181..197 /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)." /note="color: #31849b" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" /note="color: #a020f0; direction: RIGHT" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" /note="color: #ffffff; direction: RIGHT" misc_feature 337..516 /label=RAD21 homology arm 1 /note="color: #ff99cc" CDS 535..738 /codon_start=1 /label=mAID /note="note=""direct strand translation""translation=""KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNV MVSCQKSSGGPEAAAFVKVSMDGAPYLRKIDLRMYK""" /note="color: #0080ff" /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA AAFVKVSMDGAPYLRKIDLRMYK" CDS 745..1461 /codon_start=1 /product="bright green-yellow fluorescent protein derived from GFP (Lam et al., 2012)" /label=Clover /note="This feature has 3 segments: 1: 745 .. 747 / #05fd14 2: 748 .. 750 / #05fd14 / 1a 3: 751 .. 1461 / #05fd14" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" primer_bind complement(1304..1323) /label=(534) GFP seq 3 /note="color: black; sequence: GGGGCCGTCGCCGATGGGGG" polyA_signal 1582..1703 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" /note="color: #a6acb3" rep_origin complement(1710..2165) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /note="color: #ffff00" promoter 2192..2296 /gene="bla" /label=AmpR promoter /note="color: #ffffff; direction: RIGHT" promoter 2298..2655 /label=SV40 promoter /note="SV40 enhancer and early promoter" /note="color: #ffffff; direction: RIGHT" rep_origin 2506..2641 /label=SV40 ori /note="SV40 origin of replication" /note="color: #ffff00" CDS 2690..3484 /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))" /note="color: #ccffcc" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind 3147..3166 /label=(877) KanNeoR F3 /note="color: black; sequence: tcgatcaggatgatctggac" promoter complement(3783..3801) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" /note="color: #ffffff; direction: LEFT" misc_feature 3808..3982 /label=RAD21 homology arm /note="color: #ff99cc" promoter complement(4053..4071) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" /note="color: #ffffff; direction: LEFT" primer_bind complement(4078..4094) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" /note="color: #a020f0; direction: LEFT" CDS 4232..4534 /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." /note="color: #993366" /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" CDS 4883..5677 /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))" /note="color: #ccffcc" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind 5340..5359 /label=(877) KanNeoR F3 /note="color: black; sequence: tcgatcaggatgatctggac" CDS 5884..6258 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /note="color: #ccffcc" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 6396..6984 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca 301 ctagtaacgg ccgccagtgt gctggaattc gcccttgcta aaactggagc tgaatctatc 361 agtttgcttg agttatgtcg aaatacgaac agaaaacaag ctgccgcaaa gttctacagc 421 ttcttggttc ttaaaaagca gcaagctatt gagctgacac aggaagaacc gtacagtgac 481 atcatcgcaa cacctggacc aaggttccat attataggat ccggtgcagg cgccaaggag 541 aagagtgctt gtcctaaaga tccagccaaa cctccggcca aggcacaagt tgtgggatgg 601 ccaccggtga gatcataccg gaagaacgtg atggtttcct gccaaaaatc aagcggtggc 661 ccggaggcgg cggcgttcgt gaaggtatca atggacggag caccgtactt gaggaaaatc 721 gatttgagga tgtataaagc tagcatggtg agcaagggcg aggagctgtt caccggggtg 781 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc 841 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc 901 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc 961 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1021 tacgtccagg agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag 1081 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1141 gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca caacgtctat 1201 atcacggccg acaagcagaa gaacggcatc aaggctaact tcaagatccg ccacaacgtt 1261 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1321 cccgtgctgc tgcccgacaa ccactacctg agccatcagt ccaagctgag caaagacccc 1381 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gattacacat 1441 ggcatggacg agctgtacaa gtaatctaga taactgatca taatcagcca taccacattt 1501 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 1561 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 1621 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 1681 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt 1741 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 1801 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 1861 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 1921 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 1981 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 2041 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 2101 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 2161 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 2221 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 2281 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg 2341 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 2401 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2461 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 2521 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 2581 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 2641 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca 2701 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 2761 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 2821 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc 2881 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 2941 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 3001 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 3061 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 3121 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 3181 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct 3241 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 3301 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 3361 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 3421 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 3481 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 3541 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 3601 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg 3661 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca 3721 ataaaaagac agaataaaac gcacggtgtt gggtcgttgg atccggtacc cagcttttgt 3781 tccctttagt gagggttaat tgcgcgcagc tagaagcatt atagctagtg tttgattcac 3841 tagtgcttac aaattgcccc catgtgtagg ggacacagaa ccctttgaga aaacttagat 3901 ttttgtctgt acaaagtctt tgcctttttc cttcttcatt tttttccagt acattaaatt 3961 tgtcaatttc atctttgagg gaaagggcga attctgcaga tatccatcac actggcggcc 4021 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact 4081 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 4141 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 4201 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga 4261 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg 4321 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta 4381 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt 4441 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa 4501 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag 4561 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg 4621 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca 4681 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag 4741 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa 4801 ctggatggct ttctcgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga 4861 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 4921 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 4981 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 5041 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 5101 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 5161 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 5221 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 5281 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 5341 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 5401 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 5461 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 5521 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 5581 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 5641 catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct 5701 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact 5761 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 5821 tatccgctca tgagacaata accctgataa atgcttcaat aatagcacgt gaggagggcc 5881 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg 5941 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc 6001 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 6061 gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg 6121 gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag 6181 cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg 6241 gccgaggagc aggactgaca cgtgctaaaa cttcattttt aatttaaaag gatctaggtg 6301 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 6361 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 6421 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 6481 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 6541 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 6601 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 6661 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 6721 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 6781 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 6841 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 6901 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 6961 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctgggc 7021 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 7081 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 7141 gagtcagtga gcgaggaagc ggaag //