LOCUS       pT7-pelB        4088 bp    DNA     circular     12-AUG-2020
DEFINITION  
SOURCE      
  ORGANISM  
COMMENT     SUMMARY:
            empty vector; N-terminal PelB signal sequence; potential C-terminal His6; T7 promoter; like pET-20b, but additionally avoids added residues in final protein since cloning uses BseRI restriction sites
            DETAILS:
            -	Similar to pET-20b in that the vector encodes an N-terminal PelB signal sequence, but has the added advantage over pET-20b in leaving no added amino acid residues following in vivo cleavage of the PelB signal sequence by the cell's signal peptidase. This advantage is made possible by cloning into the two BseRI restriction sites.
            -	Subcloning requires ligation-independent cloning and use of the two BseRI restriction sites that are present in this empty vector. The two BseRI sites span a removable 432 nucleotide sequence. Cloning into the BseRI-digested vector can be achieved using a ligation-independent system that is dependent on homologous recombination, such as the Takara In-Fusion HD Cloning Plus system, which requires simply designing the PCR-derived insert (containing the protein of interest) to contain 15 bp at ea
            -	The sequence of the added N-terminus is the PelB signal sequence: MKYLLPTAAAGLLLLAAQPAMA.
            -	The PelB signal sequence directs the protein of interest to the E. coli inner membrane. Whether the protein traverses the inner membrane is dependent on the protein of interest. 
            -	A C-terminal His6-tag is possible: if no stop codon is included in the cloned insert, the sequence LEHHHHHH is added to the C-terminus.
            -	BseRI cuts 8-10 nucleotides outside of its own recognition sequence and is a non-palindromic site. The placement & orientation of the two BseRI sites eliminates all of the BseRI recognition sequence in the resulting subclone.
            -	To verify the PCR-generated insert sequence, use sequencing primers T7 (TAATACGACTCACTATAGGG), T7-Ter (GCTAGTTATTGCTCAGCGG).
FEATURES             Location/Qualifiers
     promoter        3348..3364
                     /vntifkey="30"
                     /label=T7\promoter
     misc_feature    3428..3493
                     /vntifkey="21"
                     /label=pelB\periplasmic\signal\sequence
     misc_feature    3932..3949
                     /vntifkey="21"
                     /label=possible\His6-tag\(if\not\stop\codon\included)
     terminator      4017..4063
                     /vntifkey="43"
                     /label=T7\terminator
     CDS             599..1456
                     /vntifkey="4"
                     /label=Amp-R
     rep_origin      12..467
                     /vntifkey="33"
                     /label=f1\origin
     CDS             complement(2648..2839)
                     /vntifkey="4"
                     /label=rop
     rep_origin      1604..2277
                     /vntifkey="33"
                     /label=origin\(ColE1)
     primer          complement(4002..4020)
                     /vntifkey="27"
                     /label=PRIMER\T7-Ter
     RBS             3411..3420
                     /vntifkey="32"
                     /label=RBS
     primer          3348..3367
                     /vntifkey="27"
                     /label=PRIMER\T7
     variation       3469..3469
                     /vntifkey="96"
                     /label=silent\mutation\C\to\T\vs.\pET-20b
     CDS             3428..3949
                     /vntifkey="4"
                     /label=expressed\protein
     misc_feature    3494..3925
                     /vntifkey="21"
                     /label=REMOVED\by\BseRI\digest;\subsequent\cloning\can\be\done\by\In-Fusion
     misc_feature    3479..3493
                     /vntifkey="21"
                     /label=add\these\15\bp,\CAGCCGGCGATGGCC,\to\the\5'\end\of\the\PCR\FORWARD\primer
     misc_feature    complement(3926..3940)
                     /vntifkey="21"
                     /label=add\these\15\bp,\GTGGTGGTGCTCGAG,\to\the\5'\end\of\the\PCR\REVERSE\primer
BASE COUNT      996 a      1056 c      1040 g       996 t 
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 
      541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 
      601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 
      661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 
      721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 
      781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 
      841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 
      901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 
      961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 
     1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 
     1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 
     1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 
     1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 
     1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 
     1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 
     1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 
     1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 
     1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 
     1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 
     1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 
     1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 
     1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 
     1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 
     1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 
     1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 
     1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 
     2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 
     2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 
     2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 
     2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 
     2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 
     2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 
     2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 
     2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 
     2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 
     2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 
     2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 
     2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 
     2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 
     2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 
     2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 
     2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 
     3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 
     3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 
     3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 
     3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 
     3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 
     3301 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 
     3361 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 
     3421 tatacatatg aaatacctgc tgccgaccgc tgctgctggt ctgctgcttc tcgctgccca 
     3481 gccggcgatg gccagatctg tcgctcctca tgtagacacc gacactgtga acaccgccat 
     3541 cctgggcgtt tcaaggtccg gcaaaggcga gacaatcgta gtgccaatga tcgacaatct 
     3601 gagccgcgca aagaaccagt ctagcatggt cgtgaacgac cccaagggcg agctctactc 
     3661 tgccagcaag gaaacacttg agaaacgcgg atacgacgtg caggtgctga acatcctgga 
     3721 tcccctgcag ggcatgagtt acaacccact ccagctcgtg atcgacgcct gggtgaacgg 
     3781 cgacgaccag gaggctgcaa agagggcaaa cacactgact ttctcactgt acaacaaccc 
     3841 aaatgctggc gacaacgctt tctttaacac cagcgcccag aatgctatca acggaatcat 
     3901 cctcgctatg aggaggcaga agcttctcga gcaccaccac caccaccact gagatccggc 
     3961 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 
     4021 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 
     4081 atccggat 
//
