测序及更多信息：https://pan.baidu.com/s/1g6n6EVX9o6hBbv9IfhXwnA 
提取码：ceee 

采购地址：淘宝店铺地址

我们的官网  http://hedgehogbio.comhttp://hedgehogbio.com/

Method for cloning

Cloning of product proteins into the intein is generally done using the unique BsrG I site (TGTACA) at the end of the intein and one of several sites downstream from the product protein (typically Hind III).  The critical detail to remember is that the intein peptide sequence must end with the amino acids: VVVHN, to be followed immediately by the initial residue of the product protein (usually methionine).  To make this possible without adding any additional residues to the product protein, we created a translationally silent restriction site at the end of the intein (BsrG I).  This site starts at nucleotide 7440 if the pET/ELP-I-CAT sequence we have sent.  Note that the BsrG I site is not in frame with the intein.  See below for a schematic of the actual upstream fusion joint:

Therefore, when you design your primers for insertion of the product protein, you must remember to add the last few nucleotides of the intein that lie between the intein’s BsrG I site and the beginning of the initial Met of the target protein (CAAC).  Also make sure that everything is in the correct frame to create a single fusion protein when the BsrG I ends are ligated.  (Note: The initial Met of the target protein is not actually involved in the cleaving reaction, and therefore might be replaced with some other amino acids.  However, this replacement will affect the structure and charge density of the cleaving junction and may have some unpredictable effects.  Our advice is to try it and see what happens.  We have also observed that the cleaving rate can be affected by the identity of the target, but have still observed cleaving in all cases.  Only the rate of cleaving is affected.)

全序列

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     CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT TTGGTGGCGG
3361     GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC GAATACCGCA AGCGACAGGC
3421     CGATCATCGT CGCGCTCCAG CGAAAGCGGT CCTCGCCGAA AATGACCCAG AGCGCTGCCG
3481     GCACCTGTCC TACGAGTTGC ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA
3541     TGCCCCGCGC CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG
3601     ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA CTGCCCGCTT
3661     TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG
3721     GCGGTTTGCG TATTGGGCGC CAGGGTGGTT TTTCTTTTCA CCAGTGAGAC GGGCAACAGC
3781     TGATTGCCCT TCACCGCCTG GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC
3841     CCCAGCAGGC GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT
3901     TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC GGACTCGGTA
3961     ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA CCAGCATCGC AGTGGGAACG
4021     ATGCCCTCAT TCAGCATTTG CATGGTTTGT TGAAAACCGG ACATGGCACT CCAGTCGCCT
4081     TCCCGTTCCG CTATCGGCTG AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA
4141     CGCAGACGCG CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC
4201     AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA AATAATACTG
4261     TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG GAACATTAGT GCAGGCAGCT
4321     TCCACAGCAA TGGCATCCTG GTCATCCAGC GGATAGTTAA TGATCAGCCC ACTGACGCGT
4381     TGCGCGAGAA GATTGTGCAC CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC
4441     GACACCACCA CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC
4501     GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA CTGTTTGCCC
4561     GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT CCGCCATCGC CGCTTCCACT
4621     TTTTCCCGCG TTTTCGCAGA AACGTGGCTG GCCTGGTTCA CCACGCGGGA AACGGTCTGA
4681     TAAGAGACAC CGGCATACTC TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC
4741     CTGAATTGAC TCTCTTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG
4801     ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA AGCAGCCCAG
4861     TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT GGTGCATGCA AGGAGATGGC
4921     GCCCAACAGT CCCCCGGCCA CGGGGCCTGC CACCATACCC ACGCCGAAAC AAGCGCTCAT
4981     GAGCCCGAAG TGGCGAGCCC GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC
5041     AACCGCACCT GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCGAGAT
5101     CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA TAACAATTCC
5161     CCTCTAGGAT CCAATAATTT TGTTTAACTT TAAGAAGGAG ATATACATAT GGGCCACGGC
5221     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
5281     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
5341     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT
5401     GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT
5461     GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC
5521     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
5581     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
5641     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT
5701     GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT
5761     GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC
5821     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
5881     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
5941     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT
6001     GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT
6061     GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC
6121     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
6181     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
6241     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT
6301     GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT
6361     GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC
6421     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
6481     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
6541     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT
6601     GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT
6661     GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC
6721     GTGGGTGTTC CGGGCGTGGG TGTTCCGGGT GGCGGTGTGC CGGGCGCAGG TGTTCCTGGT
6781     GTAGGTGTGC CGGGTGTTGG TGTGCCGGGT GTTGGTGTAC CAGGTGGCGG TGTTCCGGGT
6841     GCAGGCGTTC CGGGTGGCGG TGTGCCGGGC GGGCTGGTGA GCTCGAACAA CAACAACAAT
6901     AACAATAACA ACAACCTCGG GATCGAGGGA AGGATTTCAG AATTCGCCCT CGCAGAGGGC
6961     ACTCGGATCT TCGATCCGGT CACCGGTACA ACGCATCGCA TCGAGGATGT TGTCGGTGGG
7021     CGCAAGCCTA TTCATGTCGT GGCTGCTGCC AAGGACGGAA CGCTGCGTGC GCGGCCCGTG
7081     GTGTCCTGGT TCGACCAGGG AACGCGGGAT GTGATCGGGT TGCGGATCGC CGGTGGCGCC
7141     ATCCTGTGGG CGACACCCGA TCACAAGGTG CTGACAGAGT ACGGCTGGCG TGCCGCCGGG
7201     GAACTCCGCA AGGGAGACAG GGTGGCGCAA CCGCGACGCT TCGATGGATT CGGTGACAGT
7261     GCGCCGATTC CGGCGCGCGT GCAGGCGCTC GCGGATGCCC TGGATGACAA ATTCCTGCAC
7321     GACATGCTGG CGGAAGAACT CCGCTATTCC GTGATCCGAG AAGTGCTGCC AACGCGGCGG
7381     GCACGGACGT TCGGCCTCGA GGTCGAGGAA CTGCACACCC TCGTCGCCGA AGGGGTTGTT
7441     GTACACAACA TGAAAATCGA AGAAGGTAAA CTGGTAATCT GGATTAACGG CGATAAAGGC
7501     TATAACGGTC TCGCTGAAGT CGGTAAGAAA TTCGAGAAAG ATACCGGAAT TAAAGTCACC
7561     GTTGAGCATC CGGATAAACT GGAAGAGAAA TTCCCACAGG TTGCGGCAAC TGGCGATGGC
7621     CCTGACATTA TCTTCTGGGC ACACGACCGC TTTGGTGGCT ACGCTCAATC TGGCCTGTTG
7681     GCTGAAATCA CCCCGGACAA AGCGTTCCAG GACAAGCTGT ATCCGTTTAC CTGGGATGCC
7741     GTACGTTACA ACGGCAAGCT GATTGCTTAC CCGATCGCTG TTGAAGCGTT ATCGCTGATT
7801     TATAACAAAG ATCTGCTGCC GAACCCGCCA AAAACCTGGG AAGAGATCCC GGCGCTGGAT
7861     AAAGAACTGA AAGCGAAAGG TAAGAGCGCG CTGATGTTCA ACCTGCAAGA ACCGTACTTC
7921     ACCTGGCCGC TGATTGCTGC TGACGGGGGT TATGCGTTCA AGTATGAAAA CGGCAAGTAC
7981     GACATTAAAG ACGTGGGCGT GGATAACGCT GGCGCGAAAG CGGGTCTGAC CTTCCTGGTT
8041     GACCTGATTA AAAACAAACA CATGAATGCA GACACCGATT ACTCCATCGC AGAAGCTGCC
8101     TTTAATAAAG GCGAAACAGC GATGACCATC AACGGCCCGT GGGCATGGTC CAACATCGAC
8161     ACCAGCAAAG TGAATTATGG TGTAACGGTA CTGCCGACCT TCAAGGGTCA ACCATCCAAA
8221     CCGTTCGTTG GCGTGCTGAG CGCAGGTATT AACGCCGCCA GTCCGAACAA AGAGCTGGCA
8281     AAAGAGTTCC TCGAAAACTA TCTGCTGACT GATGAAGGTC TGGAAGCGGT TAATAAAGAC
8341     AAACCGCTGG GTGCCGTAGC GCTGAAGTCT TACGAGGAAG AGTTGGCGAA AGATCCACGT
8401     ATTGCCGCCA CCATGGAAAA CGCCCAGAAA GGTGAAATCA TGCCGAACAT CCCGCAGATG
8461     TCCGCTTTCT GGTATGCCGT GTGTACTGCG GTGATCAACG CCGCCAGCGG TCGTCAGACT
8521     GTCGATGAAG CCCTGAAAGA CGCGCAGACT AATTCGAGCT CGTGACTGCA GGCAAGCTTG
8581     CGGCCGCACT CGAGCACCAC CACCACCACC ACTGAGATCC GGCTGCTAAC AAAGCCCGAA
8641     AGGAAGCTGA GTTGGCTGCT GCCACCGCTG AGCAATAACT AGCATAACCC CTTGGGGCCT
8701     CTAAACGGGT CTTGAGGGGT TTTTTGCTGA AAGGAGGAAC TATATCCGGA T