Below are the results of PARTIAL sequencing of pLEfR1FHrT obtained from Genentech. Ref: Kim, HS et al., "Translation levels control multi-spanning membrane protein expression," PLoS One. 2012;7(4):e35844. tphac promoter; 79 amino acid LE leader; EG-VEGFR1 gene is cloned between BamHI/NotI sites. There's a thrombin site after the LE leader and EG-VEGFR1 has a flag tag + octaHis tag at the CT. Intact plasmid is ~6kb (based on agarose gel) and according to the paper, contains the tRNAs for 3 rare E. coli codons (argU, glyT and pro2). AmpR 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 AhdI ------+----- PvuII NmeAIII ----+-- ------- 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 >>.......LE leader.........> ttcactggac agagatctcg acagccgtat tgaactggaa atgcgtaccg atcataaaga gctgtctgaa catctgatgc g s l d r d l d s r i e l e m r t d h k e l s e h l m >......................................LE leader......................................> tggttgatct cgcccgtaat gatctggcac gcatttgcac ccccggcagc cgctacgtcg ccgatctcac caaagttgac l v d l a r n d l a r i c t p g s r y v a d l t k v d >......................................LE leader......................................> BamHI -+---- cgttattcct atgtgatgca cctcgtctct cgcgtagtcg gcgaactgcg tcttgtcccc cgtggatcca tggagaccac r y s y v m h l v s r v v g e l r l v p r g s m e t >......................LE leader......................>> >>.Thrombin site.>> EG-VEGFR1 >>.........> catggggttc atggatgaca atgccaccaa cacttccacc agcttccttt ctgtgctcaa ccctcatgga gcccatgcca t m g f m d d n a t n t s t s f l s v l n p h g a h a >......................................EG-VEGFR1......................................> SfcI -+---- AcuI ------- Eco57MI PfoI ------- -+----- cttccttccc attcaacttc agctacagcg actatgatat gcctttggat gaagatgagg atgtgaccaa ttccaggacg t s f p f n f s y s d y d m p l d e d e d v t n s r t >......................................EG-VEGFR1......................................> ttctttgctg ccaagattgt cattgggatg gccctggtgg gcatcatgct ggtctgcggc attggaaact tcatctttat f f a a k i v i g m a l v g i m l v c g i g n f i f >......................................EG-VEGFR1......................................> cgctgccctg gtccgctaca agaaactgcg caacctcacc aacctgctca tcgccaacct ggccatctct gacttcctgg i a a l v r y k k l r n l t n l l i a n l a i s d f l >......................................EG-VEGFR1......................................> BmgBI ApaLI ----+-- -+---- tggccattgt ctgctgcccc cttgagatgg actactatgt ggtgcgccag ctctcctggg agcacggcca cgtcctgtgc v a i v c c p l e m d y y v v r q l s w e h g h v l c >......................................EG-VEGFR1......................................> acctctgtca actacctgcg cactgtctct ctctatgtct ccaccaatgc cctgctggcc atcgccattg acaggtatct t s v n y l r t v s l y v s t n a l l a i a i d r y >......................................EG-VEGFR1......................................> AlwNI ------+-- ggctattgtc catccgctga gaccacggat gaagtgccaa acagccactg gcctgattgc cttggtgtgg acggtgtcca l a i v h p l r p r m k c q t a t g l i a l v w t v s >......................................EG-VEGFR1......................................> BsaBI EciI -----+----- ------- tcctgatcgc catcccttcc gcctacttca ccaccgagac ggtcctcgtc attgtcaaga gccaggaaaa gatcttctgc i l i a i p s a y f t t e t v l v i v k s q e k i f c >......................................EG-VEGFR1......................................> ApaI -----+ ApoI EcoO109I -+----- --+---- EcoRI PspOMI -+----- -+---- ggccagatct ggcctgtgga ccagcagctc tactacaagt cctacttcct ctttatcttt ggcatagaat tcgtgggccc g q i w p v d q q l y y k s y f l f i f g i e f v g >......................................EG-VEGFR1......................................> BstEII -+----- cgtggtcacc atgaccctgt gctatgccag gatctcccgg gagctctggt tcaaggcggt ccctggattc cagacagagc p v v t m t l c y a r i s r e l w f k a v p g f q t e >......................................EG-VEGFR1......................................> GsaI -----+ BbsI BseYI ------- -+---- agatccgcaa gaggctgcgc tgccgcagga agacggtcct ggtgctcatg tgcatcctca ccgcctacgt gctatgctgg q i r k r l r c r r k t v l v l m c i l t a y v l c w >......................................EG-VEGFR1......................................> HaeII -----+ SfoI ---+-- BsaHI --+--- NarI --+--- KasI AleI BtsI -+---- ------+---- ------ gcgcccttct acggcttcac catcgtgcgc gatttcttcc ccaccgtgtt cgtgaaggag aagcactacc tcactgcctt a p f y g f t i v r d f f p t v f v k e k h y l t a >......................................EG-VEGFR1......................................> DraIII ------+--- BclI -+----- ctacatcgtc gagtgcatcg ccatgagcaa cagcatgatc aacactctgt gcttcgtgac cgtcaagaac gacaccgtca f y i v e c i a m s n s m i n t l c f v t v k n d t v >......................................EG-VEGFR1......................................> ScaI ---+-- TatI BsgI -+---- ------ agtacttcaa aaagatcatg ttgctccact ggaaggcttc ttacaatggc ggtaagtcca gtgcagacct ggacctcaag k y f k k i m l l h w k a s y n g g k s s a d l d l k >......................................EG-VEGFR1......................................> EarI ------ acagttggga tgcctgccac cgaagaggtg gactgcatca gactaaaaga ctacaaggat gacgatgaca agcaccatca t v g m p a t e e v d c i r l k d y k d d d d k h h >....................EG-VEGFR1....................>> >>.......Flag tag........>> octaHis tag >>.....> PspXI --+------ XhoI KpnI -+----- ------+ NotI Acc65I --+----- -+----- ccatcaccat caccattaaa tgcggccgct cgagctcggt acccggggat ctaggcctaa cgctcggttg ccgccgggcg h h h h h h - m r p l e l g t r g s r p n a r l p p g >..octaHis tag.>> ttttttattg ttaatcagtt agcgaaatat cttacttgca atcggtgtgg aaaacggtag tattagcagc cacgagtcgg v f y c - s v s e i s y l q s v w k t v v l a a t s r MmeI ------ cacgtagcgc agcctggtag cgcaccgtca tggggtgtcg ggggtcggag ttcaaatcct ctcgtgccga ccaaaaatcc h v a q p g s a p s w g v g g r s s n p l v p t k n AccI ---+--- AseI SalI --+--- --+---- caagaaaaaa ccaaccctta cggttggttt ttttatatct gcaattaatt cgataaacag accgtgacag tcgacagatc p 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 tctcatgttt gacag l s c l t