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pTop1 vector (V002572#)

Basic Vector Information

Basic Vector Information
Vector Name pTop1 Antibiotic Resistance Ampicillin
Length 10745 bp Type Chloroplast transformation vector
Source Lu Y, Rijzaani H, Karcher D, Ruf S, Bock R.

pTop1 vector Vector Map

tRNA-Met ribosomal protein S14 psaB T7 promoter M13 fwd f1 ori AmpR promoter AmpR CAP binding site lac promoter lac operator M13 rev T3 promoter psbZ tRNA-Gly LoxP prrN promoter aadA psbA terminator LoxP KS primer rbcL terminator HPT IGR2 TCY IGR1 TMT prrN promoter T3 promoter

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

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               10745 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Chloroplast transformation vector pTop1, complete sequence.
ACCESSION   JX235345
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10745
                     /organism="Chloroplast transformation vector pTop1"
                     /mol_type="other DNA"
                     /db_xref="taxon:1229083"
     tRNA            complement(31..104)
                     /product="tRNA-Met"
     CDS             complement(254..556)
                     /codon_start=1
                     /product="ribosomal protein S14"
                     /label=ribosomal protein S14
                     /protein_id="AFQ99317.1"
                     /translation="MARKSLIQREKKRQKLEQKYHSIRRSSKKEISKVPSLSDKWEIYG
                     KLQSPPRNSAPTRLHRRCFLTGRPRANYRDFGLSGHILREMVHACLLPGATRSSW"
     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="AFQ99318.1"
                     /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="AFQ99319.1"
                     /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(6897..7117)
                     /regulatory_class="terminator"
                     /note="rbcL terminator; derived from Nicotiana tabacum"
     CDS             complement(7118..8044)
                     /codon_start=1
                     /gene="HPT"
                     /product="homogentisate phytyl transferase"
                     /label=HPT
                     /note="derived from Synechocystis sp. PCC 6803"
                     /protein_id="AFQ99320.1"
                     /translation="MATIQAFWRFSRPHTIIGTTLSVWAVYLLTILGDGNSVNSPASLD
                     LVFGAWLACLLGNVYIVGLNQLWDVDIDRINKPNLPLANGDFSIAQGRWIVGLCGVASL
                     AIAWGLGLWLGLTVGISLIIGTAYSVPPVRLKRFSLLAALCILTVRGIVVNLGLFLFFR
                     IGLGYPPTLITPIWVLTLFILVFTVAIAIFKDVPDMEGDRQFKIQTLTLQIGKQNVFRG
                     TLILLTGCYLAMAIWGLWAAMPLNTAFLIVSHLCLLALLWWRSRDVHLESKTEIASFYQ
                     FIWKLFFLEYLLYPLALWLPNFSNTIF"
     gene            complement(7118..8044)
                     /gene="HPT"
                     /label=HPT
                     /note="SyHPT"
     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"
     regulatory      complement(10528..10679)
                     /regulatory_class="promoter"
                     /note="prrN promoter; derived from Nicotiana tabacum"
     RBS             10535..10557
                     /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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