LOCUS Exported 4043 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION BIOFAB reporter plasmid for measuring T3 early termination efficiency. ACCESSION . VERSION . KEYWORDS pFAB804 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4043) AUTHORS Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D TITLE Precise and reliable gene expression via standard transcription and translation initiation elements. JOURNAL Nat Methods. 2013 Apr;10(4):354-60. doi: 10.1038/nmeth.2404. Epub 2013 Mar 10. PUBMED 23474465 REFERENCE 2 (bases 1 to 4043) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4043 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 320..393 /label=PLtetO-1 promoter /note="modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)" protein_bind 320..338 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 345..363 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" RBS 407..418 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 425..1102 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" terminator 1206..1235 /label=T3Te terminator /note="phage T3 early transcription terminator" RBS 1352..1363 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 1370..2083 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" primer_bind complement(1400..1428) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 2023..2043 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" terminator 2119..2190 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2206..2233 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(2304..2321) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2438..2983) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(2518..2537) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 3097..3191 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(3222..4016) /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3333..3352) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 3943..3962 /label=Neo-R /note="Neomycin resistance gene, reverse primer" ORIGIN 1 atcagatcat gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 61 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg acgtctaggg 121 ataacagggt aattacggcc ccagaattca aaagatctta agtaagtaag agtatacgta 181 tatcggctaa aacgtattaa ggcgcttcgg cgcctttttt tatgggggta ttttcatccc 241 aatccacacg tccaacgcac agcaaacacc acgtcgaccc tatcagctgc gtgctttcta 301 tgagtcgttg ctgcatatct ccctatcagt gatagagatt gacatcccta tcagtgatag 361 agatactgag cacatcagca ggacgcactg acctttggaa ttcattaaag aggagaaagg 421 taccatggcg agtagcgaag acgttatcaa agagttcatg cgtttcaaag ttcgtatgga 481 aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga 541 aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt tcgcttggga 601 catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc cggctgacat 661 cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt 721 cgaagacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg gtgagttcat 781 ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta tgcagaaaaa 841 aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg 901 tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg aagttaaaac 961 cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg acatcaaact 1021 ggacatcacc tcccacaacg aagactacac catcgttgaa cagtacgaac gtgctgaagg 1081 tcgtcactcc accggtgctt aataatctgc tgagtgatag actcaaggtc gctcctagcg 1141 agtggccttt atgattatca ctttacttat gtgataccct caagagaaaa tgtaaccaac 1201 tcactggctc accttcacgg gtgggccttt cttcgttccg ggcattaacc ctcactaaca 1261 ggagaggcgg actcactata gagggacaaa ctcaaggtca ttcgcaagag tggcctttat 1321 gattgacctt cttccggtta atcaattcat taaagaggag aaaggtacca tgagcaaagg 1381 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg 1441 gcacaaattt tctgtccgtg gagagggtga aggtgatgct acaaacggaa aactcaccct 1501 taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg tcactactct 1561 gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc atgacttttt 1621 caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca aagatgacgg 1681 gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta atcgtatcga 1741 gttaaagggt attgatttta aagaagatgg aaacattctt ggacacaaac tcgagtacaa 1801 ctttaactca cacaatgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa 1861 cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc attatcaaca 1921 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtcgacaca 1981 atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt 2041 aactgctgct gggattacac atggcatgga tgagctctac aaataaggat cggttgtcga 2101 gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 2161 tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt 2221 gggcctttct gcgtttatag gatcctaact cgagctgaag ccaataggga taacagggta 2281 atcctaggga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg 2341 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata 2401 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg 2461 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa 2521 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt 2581 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt 2641 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac 2701 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca 2761 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg 2821 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg 2881 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc 2941 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa 3001 tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt 3061 cagccccata cgatataagt tgttactagt gcttggattc tcaccaataa aaaacgcccg 3121 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat 3181 caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga 3241 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag 3301 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc 3361 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt 3421 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg 3481 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg 3541 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga 3601 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt 3661 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc 3721 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca 3781 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt 3841 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac 3901 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat 3961 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga 4021 aacgatcctc atcctgtctc ttg //