Below are the results of PARTIAL sequencing of p20LEfCD20rT obtained from Genentech. This vector was used to express eukaryotic membrane proteins in E. coli. Ref: Kim, HS et al., "Translation levels control multi-spanning membrane protein expression," PLoS One. 2012;7(4):e35844. p20LEfCD20rT: AmpR tphac promoter 20 amino acid trp LE leader CD20 gene CT octaHis tag Plasmid is ~5.5kb and contains the tRNAs for 3 rare E. coli codons (argU, glyT and pro2). BamHI/NotI digestion of the plasmid gave a dropout of the correct size by agarose gel. BamHI & NotI sites appear to be unique and can be used for restriction digest subcloning. Alternatively, you can use SLIC cloning methods and liberalize the vector via PCR. I do not know if the other sites listed are unique. The sequencing revealed a thrombin site inserted after residue 9 of CD20 (not sure why this is there). NcoI -+---- HpaI SnaBI ----+-- ---+-- aggcctaacg ctcggttgcc gccgggcgtt ttttattgtt aaccatggag cgattacgta aagaagttat tgaagcatcc g l t l g c r r a f f i v n h g a i t - r s y - s i >>..............................>> Lambda transcriptional terminator PvuII AhdI ----+-- ------+----- tcgtcagtaa aaagttaatc ttttcaacag ctgtcataaa gttgtcacgg ccgagactta tagtcgcttt aattgtgagc l v s k k l i f s t a v i k l s r p r l i v a l i v s >>....PhoA -10.....>> Pho Box >>...>> lac operator >>.......> SpeI XbaI -+----- -+----- ggataacaat ttgtaactag tacgcaagtt cacgtaaaaa gggtatctag aattatgaaa gcaattttcg tactgaaagg g - q f v t s t q v h v k r v s r i m k a i f v l k >.........>> lac operator >>......20 LE leader.......> BamHI -+---- ttcactggac agagatctcg acagccgtat tgaaggatcc atgacaacac ccagaaattc agtaaatctt gtcccccgtg g s l d r d l d s r i e g s m t t p r n s v n l v p r >..............20 LE leader..............>> >>.................CD20...................> >>thrombin ..> BtsI ------ gcagtgggac tttcccggca gagccaatga aaggccctat tgctatgcaa tctggtccaa aaccactctt ccgtcgcatg g s g t f p a e p m k g p i a m q s g p k p l f r r m >........................................CD20.........................................> >..>> thrombin ApaI -----+ BaeGI -----+ PspOMI HindIII BspHI -+---- -+---- -+---- tcctccttgg tgggccccac gcaaagcttc ttcatgaggg aatctaagac tttgggggct gtccagatta tgaatgggct s s l v g p t q s f f m r e s k t l g a v q i m n g >........................................CD20.........................................> GsaI -----+ SapI PasI BseYI BtgZI ------- --+---- -+---- ------ cttccacatt gccctggggg gtcttctgat gatcccagca gggatctatg cacccatcgc tgtgactgtg tggtaccctc l f h i a l g g l l m i p a g i y a p i a v t v w y p >........................................CD20.........................................> BsaWI -+----- BspEI -+----- tctggggcgg catcatgtat attatttccg gatcactcct ggcagcaacg gagaaaaact ccaggaagag tttggtcaaa l w g g i m y i i s g s l l a a t e k n s r k s l v k >........................................CD20.........................................> EcoRI SspI -+---- ---+-- ggaaaaatga taatgaattc attgagcctc tttgctgcca tttctggaat gattctttca atcatggaca tacttaatat g k m i m n s l s l f a a i s g m i l s i m d i l n >........................................CD20.........................................> DraI ---+--- taaaatttcc cattttttaa aaatggagag tctgaatttt attagagctc acacaccata tattaacata tacaactgtg i k i s h f l k m e s l n f i r a h t p y i n i y n c >........................................CD20.........................................> aaccagctaa tccctctgag aaaaactccc catctaccca atactgttac agcatacaat ctctgttctt gggcattttg e p a n p s e k n s p s t q y c y s i q s l f l g i l >........................................CD20.........................................> BsaBI ------+---- tcagtgatgc tgatctttgc cttcttccag gaacttgtaa tagctggcat cgttgagaat gaatggaaaa gaacgagctc s v m l i f a f f q e l v i a g i v e n e w k r t s >........................................CD20.........................................> cagacccaaa tctaacatag ttctcctgtc agcagaagaa aaaaaagaac agactattga aataaaagaa gaagtggttg s r p k s n i v l l s a e e k k e q t i e i k e e v v >........................................CD20.........................................> XmnI -----+----- ggctaactga aacatcttcc caaccaaaga atgaagaaga cattgaaatt attccaatcc aagaagagga agaagaagaa g l t e t s s q p k n e e d i e i i p i q e e e e e e >........................................CD20.........................................> BsmBI BseRI ------ ------ acagagacga actttccaga acctccccaa gatcaggaat cctcaccaat agaaaatgac agctctcctc accaccacca t e t n f p e p p q d q e s s p i e n d s s p h h h >..................................CD20..................................>> >>His tag..> PspXI --+------ SmlI -+---- XhoI SmaI -+---- ----+-- NotI XmaI --+------ -+----- ccaccaccac cactaaagcg gccgctcgag ctcggtaccc ggggatctag gcctaacgct cggttgccgc cgggcgtttt h h h h h - s g r s s s v p g d l g l t l g c r r a f >..His tag..>> ttattgttaa tcagttagcg aaatatctta cttgcaatcg gtgtggaaaa cggtagtatt agcagccacg agtcggcacg f i v n q l a k y l t c n r c g k r - y - q p r v g t tagcgcagcc tggtagcgca ccgtcatggg gtgtcggggg tcggaggttc aaatcctctc gtgccgacca aaaatcccaa - r s l v a h r h g v s g v g g s n p l v p t k n p AccI --+---- AseI SalI --+--- -+----- gaaaaaacca acccttacgg ttggtttttt tatatctgca attaattcga taaacagacc gtgacagtcg acagatctct k k k p t l t v g f f i s a i n s i n r p - q s t d l ClaI ---+--- catgtttgac agcttatcat cgatagatca aaagccattg actcagcaag ggttgaccgt ataattcacg cgattacacc s c l t a y h r - i k s h - l s k g - p y n s r d y t BstBI --+--- BspMI SfcI ------- -+---- gcattgcggt atcaacgcgc ccttagctca gttggataga gcaacgacct tctaagtcgt gggccgcagg ttcgaatcct a l r y q r a l s s v g - s n d l l s r g p q v r i AscI --+------ BssHII -+----- PstI -----+ SbfI MunI BbvCI ------+- -+----- --+----- gcagggcgcg ccattacaat tggtagccga gttccaggat gcgggcatcg tataatggct attacctcag ccttccaagc l q g a p l q l v a e f q d a g i v - w l l p q p s k tgatgatgcg ggttcgattc ccgctgcccg ctccaagatg tgacatattt gggacattat caccaaaaat gtcgtctatt l m m r v r f p l p a p r c d i f g t l s p k m s s i SphI -----+ ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact act l h a p f l a a a v l n g l n l l