Basic Vector Information
pTop1 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pTop1 vector Sequence
LOCUS 40924_43778 10745 bp DNA circular SYN 18-DEC-2018 DEFINITION Chloroplast transformation vector pTop1, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10745) AUTHORS Lu Y, Rijzaani H, Karcher D, Ruf S, Bock R. TITLE Efficient engineering of the vitamin E metabolic pathway in transgenic tobacco and tomato plastids with synthetic multigene operons JOURNAL Unpublished REFERENCE 2 (bases 1 to 10745) AUTHORS Lu Y, Rijzaani H, Karcher D, Ruf S, Bock R. TITLE Direct Submission JOURNAL Submitted (25-JUN-2012) Organelle Biology, Biotechnology and Molecular Ecophysiology, Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, Potsdam-Golm, Brandenburg 14476, Germany REFERENCE 3 (bases 1 to 10745) TITLE Direct Submission REFERENCE 4 (bases 1 to 10745) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Unpublished" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (25-JUN-2012) Organelle Biology, Biotechnology and Molecular Ecophysiology, Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, Potsdam-Golm, Brandenburg 14476, Germany" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..10745 /mol_type="other DNA" /organism="synthetic DNA construct" tRNA complement(31..104) /product="tRNA-Met" CDS complement(257..556) /gene="rps14" /label=rps14 /note="Small ribosomal subunit protein uS14c from Nicotiana sylvestris. Accession#: Q3C1I0" gene complement(679..1878) /gene="psaB" /label=psaB /note="derived from Nicotiana tabacum" promoter complement(1891..1909) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1919..1935) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 2077..2532 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 2558..2662 /label=AmpR promoter CDS 2663..3520 /label=AmpR /note="beta-lactamase" rep_origin 3694..4282 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 4570..4591 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 4606..4636 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4644..4660 /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)." primer_bind 4668..4684 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 4705..4723 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" gene 4736..4867 /gene="psbZ" /label=psbZ /note="derived from Nicotiana tabacum" tRNA 5143..5213 /product="tRNA-Gly" protein_bind 5406..5439 /label=loxP /note="Cre-mediated recombination occurs in the 8-bp core sequence (ATGTATGC) (Shaw et al., 2021)." regulatory 5480..5614 /note="prrN promoter; derived from Nicotiana tabacum" /regulatory_class="promoter" CDS 5615..6403 /label=SmR /note="aminoglycoside adenylyltransferase (Murphy, 1985)" regulatory 6417..6811 /note="psbA terminator; derived from Nicotiana tabacum" /regulatory_class="terminator" misc_recomb 6815..6848 /label=LoxP /note="LoxP" protein_bind complement(6815..6848) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 6878..6894 /label=KS primer /note="common sequencing primer, one of multiple similar variants" regulatory complement(6897..7117) /note="rbcL terminator; derived from Nicotiana tabacum" /regulatory_class="terminator" CDS complement(7121..8044) /note="Homogentisate phytyltransferase from Synechocystis sp. (strain PCC 6803 / Kazusa). Accession#: P73726" misc_feature complement(8045..8291) /note="IGR2; intergenic region between Nicotiana tabacum plastid rps2 and atpI" CDS complement(8324..9415) /codon_start=1 /gene="TCY" /product="tocopherol cyclase" /label=TCY /note="derived from Synechocystis sp. PCC 6803" /protein_id="AFQ99321.1" /translation="MKFPPHSGYHWQGQSPFFEGWYVRLLLPQSGESFAFMYSIENPAS DHHYGGGAVQILGPATKKQENQEDQLVWRTFPSVKKFWASPRQFALGHWGKCRDNRQAK PLLSEEFFATVKEGYQIHQNQHQGQIIHGDRHCRWQFTVEPEVTWGSPNRFPRATAGWL SFLPLFDPGWQILLAQGRAHGWLKWQREQYEFDHALVYAEKNWGHSFPSRWFWLQANYF PDHPGLSVTAAGGERIVLGRPEEVALIGLHHQGNFYEFGPGHGTVTWQVAPWGRWQLKA SNDRYWVKLSGKTDKKGSLVHTPTAQGLQLNCRDTTRGYLYLQLGSVGHGLIVQGETDT AGLEVGGDWGLTEENLSKKTVPF" gene complement(8324..9415) /gene="TCY" /label=TCY /note="SyTCY" misc_feature complement(9416..9564) /note="IGR1; intergenic region between Nicotiana tabacum plastid psbH and petB" CDS complement(9574..10527) /codon_start=1 /gene="TMT" /product="gamma-tocopherol methyltransferase" /label=TMT /note="derived from Synechocystis sp. PCC 6803" /protein_id="AFQ99322.1" /translation="MVYHVRPKHALFLAFYCYFSLLTMASATIASADLYEKIKNFYDDS SGLWEDVWGEHMHHGYYGPHGTYRIDRRQAQIDLIKELLAWAVPQNSAKPRKILDLGCG IGGSSLYLAQQHQAEVMGASLSPVQVERAGERARALGLGSTCQFQVANALDLPFASDSF DWVWSLESGEHMPNKAQFLQEAWRVLKPGGRLILATWCHRPIDPGNGPLTADERRHLQA IYDVYCLPYVVSLPDYEAIARECGFGEIKTADWSVAVAPFWDRVIESAFDPRVLWALGQ AGPKIINAALCLRLMKWGYERGLVRFGLLTGIKPLV" gene complement(9574..10527) /gene="TMT" /label=TMT /note="SyTMT" RBS complement(10535..10557) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" promoter complement(10692..10710) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase"
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