Basic Vector Information
pTop2 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 ℃.
pTop2 vector Sequence
LOCUS Exported 10829 bp ds-DNA circular SYN 18-DEC-2018
DEFINITION Chloroplast transformation vector pTop2, complete sequence.
ACCESSION JX235346
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10829)
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JOURNAL Unpublished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 10829)
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 10829)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10829
/organism="Chloroplast transformation vector pTop2"
/mol_type="other DNA"
/db_xref="taxon:1229084"
tRNA complement(31..104)
/product="tRNA-Met"
CDS complement(254..556)
/codon_start=1
/gene="rps14"
/product="ribosomal protein S14"
/label=rps14
/protein_id="AFQ99323.1"
/translation="MARKSLIQREKKRQKLEQKYHSIRRSSKKEISKVPSLSDKWEIYG
KLQSPPRNSAPTRLHRRCFLTGRPRANYRDFGLSGHILREMVHACLLPGATRSSW"
gene complement(254..556)
/gene="rps14"
/label=rps14
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
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2558..2662
/gene="bla"
/label=AmpR promoter
CDS 2663..3523
/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"
CDS 2861..3523
/codon_start=1
/gene="ampR"
/product="beta-lactamase"
/label=ampR
/protein_id="AFQ99324.1"
/translation="MSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHL
TDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEA
IPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFI
ADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
gene 2861..3523
/gene="ampR"
/label=ampR
rep_origin 3618..4300
rep_origin 3694..4282
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 4570..4591
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/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"
misc_recomb 5406..5439
/label=LoxP
/note="LoxP"
protein_bind complement(5406..5439)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
regulatory 5480..5614
/regulatory_class="promoter"
/note="prrN promoter; derived from Nicotiana tabacum"
CDS 5615..6406
/codon_start=1
/gene="aadA"
/product="streptomycin/spectinomycin adenylyltransferase"
/label=aadA
/protein_id="AFQ99325.1"
/translation="MAEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVLEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
gene 5615..6406
/gene="aadA"
/label=aadA
regulatory 6417..6811
/regulatory_class="terminator"
/note="psbA terminator; derived from Nicotiana tabacum"
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(6932..7153)
/regulatory_class="terminator"
/note="rbcL terminator; derived from Chlamydomonas
reinhardtii"
CDS complement(7160..8068)
/codon_start=1
/gene="TMT"
/product="gamma-tocopherol methyltransferase"
/label=TMT
/note="derived from Arabidopsis thaliana"
/protein_id="AFQ99326.1"
/translation="MVAVAAAATSTEALRKGIAEFYNETSGLWEEIWGDHMHHGFYDPD
SSVQLSDSGHKEAQIRMIEESLRFAGVTDEEEEKKIKKVVDVGCGIGGSSRYLASKFGA
ECIGITLSPVQAKRANDLAAAQSLAHKASFQVADALDQPFEDGKFDLVWSMESGEHMPD
KAKFVKELVRVAAPGGRIIIVTWCHRNLSAGEEALQPWEQNILDKICKTFYLPAWCSTD
DYVNLLQSHSLQDIKCADWSENVAPFWPAVIRTALTWKGLVSLLRSGMKSIKGALTMPL
MIEGYKKGVIKFGIITCQKPL"
gene complement(7160..8068)
/gene="TMT"
/label=TMT
/note="AtTMT"
regulatory 8061..8070
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
stem_loop complement(8088..8137)
/note="IEE; intercistronic expression element"
regulatory complement(8150..8299)
/regulatory_class="terminator"
/note="rps16 terminator; derived from Nicotiana tabacum"
CDS complement(8300..9391)
/codon_start=1
/gene="TCY"
/product="tocopherol cyclase"
/label=TCY
/note="derived from Synechocystis sp. PCC 6803"
/protein_id="AFQ99327.1"
/translation="MKFPPHSGYHWQGQSPFFEGWYVRLLLPQSGESFAFMYSIENPAS
DHHYGGGAVQILGPATKKQENQEDQLVWRTFPSVKKFWASPRQFALGHWGKCRDNRQAK
PLLSEEFFATVKEGYQIHQNQHQGQIIHGDRHCRWQFTVEPEVTWGSPNRFPRATAGWL
SFLPLFDPGWQILLAQGRAHGWLKWQREQYEFDHALVYAEKNWGHSFPSRWFWLQANYF
PDHPGLSVTAAGGERIVLGRPEEVALIGLHHQGNFYEFGPGHGTVTWQVAPWGRWQLKA
SNDRYWVKLSGKTDKKGSLVHTPTAQGLQLNCRDTTRGYLYLQLGSVGHGLIVQGETDT
AGLEVGGDWGLTEENLSKKTVPF"
gene complement(8300..9391)
/gene="TCY"
/label=TCY
/note="SyTCY"
stem_loop complement(8409..9458)
/note="IEE; intercistronic expression element"
regulatory complement(9464..9684)
/regulatory_class="terminator"
/note="rbcL terminator; derived from Nicotiana tabacum"
CDS complement(9685..10611)
/codon_start=1
/gene="HPT"
/product="homogentisate phytyl transferase"
/label=HPT
/note="derived from Synechocystis sp. PCC 6803"
/protein_id="AFQ99328.1"
/translation="MATIQAFWRFSRPHTIIGTTLSVWAVYLLTILGDGNSVNSPASLD
LVFGAWLACLLGNVYIVGLNQLWDVDIDRINKPNLPLANGDFSIAQGRWIVGLCGVASL
AIAWGLGLWLGLTVGISLIIGTAYSVPPVRLKRFSLLAALCILTVRGIVVNLGLFLFFR
IGLGYPPTLITPIWVLTLFILVFTVAIAIFKDVPDMEGDRQFKIQTLTLQIGKQNVFRG
TLILLTGCYLAMAIWGLWAAMPLNTAFLIVSHLCLLALLWWRSRDVHLESKTEIASFYQ
FIWKLFFLEYLLYPLALWLPNFSNTIF"
gene complement(9685..10611)
/gene="HPT"
/label=HPT
/note="SyHPT"
regulatory complement(10612..10763)
/regulatory_class="promoter"
/note="prrN promoter; derived from Nicotiana tabacum"
RBS 10619..10641
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
promoter complement(10776..10794)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
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