LOCUS       pMBP-HsSyx-375-792        7167 bp    DNA     circular SYN 17-NOV-2021
DEFINITION  
SOURCE      
  ORGANISM  
COMMENT     SUMMARY:
            E. coli expression clone (T7 promoter) for expression-optimized DNA of human 
            Syx (aa 375-792) with N-terminal His6 + mature maltose binding protein + 
            TEV protease cleavage site.
            DETAILS:
            -	Protein sequence is MKSS + HHHHHH + GSS + maltose binding protein 
            (aa 27-393 of AAB59056) + NGIE + TEV protease cleavage (ENLYFQ*S; * = cleavage 
            site) + N + human Syx (synectin-binding guanine exchange factor; aa 375-792 from 
            accession number NP_001036128.1; DNA for residues 375-392 was generated by 
            oligonucleotides using optimal codons).
            -	Confers ampicillin-resistance.
            -	Backbone vector is pET His6 MBP TEV LIC (2M-T) Addgene-29708.
            Construct design by Ryan Boyd and Debra T. Hansen. 
            Cloning by Felicia M. Craciunescu.
FEATURES             Location/Qualifiers
     promoter        104..208
                     /gene="bla"
                     /vntifkey="30"
                     /label=AmpR\promoter
     CDS             209..1069
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /vntifkey="4"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and related antibiotics"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /vntifkey="33"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     misc_feature    2014..2156
                     /vntifkey="21"
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /vntifkey="4"
                     /label=rop
     promoter        4010..4028
                     /vntifkey="30"
                     /label=T7\promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /vntifkey="32"
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /vntifkey="4"
                     /label=6xHis
     CDS             4186..5286
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /vntifkey="4"
                     /label=MBP
                     /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol."
     CDS             5299..5319
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /vntifkey="4"
                     /label=TEV\site
     terminator      6717..6764
                     /vntifkey="43"
                     /label=T7\terminator
                     /note="transcription terminator for bacteriophage T7 RNA polymerase"
     promoter        complement(7130..7158)
                     /vntifkey="30"
                     /label=tet\promoter
                     /note="E. coli promoter for tetracycline efflux protein gene"
     CDS             5377..6576
                     /vntifkey="4"
                     /label=HsSyx\aa393-792,\expression-optimized
     primer          4010..4029
                     /vntifkey="27"
                     /label=PRIMER\T7
     primer          complement(6703..6721)
                     /vntifkey="27"
                     /label=PRIMER\T7TERM
     primer          5163..5187
                     /vntifkey="27"
                     /label=PRIMER\MBPpelBseqF
     CDS             4147..6576
                     /vntifkey="4"
                     /label=EXPRESSED\PROTEIN
     primer          5193..5213
                     /vntifkey="27"
                     /label=PRIMER\MBPFOR
     primer          4034..4055
                     /vntifkey="27"
                     /label=PRIMER\37237\seq\F
     CDS             5323..5376
                     /vntifkey="4"
                     /label=HsSyx\aa375-392,\codon-optimized
BASE COUNT     1760 a      1828 c      2020 g      1559 t 
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 
     3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 
     3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 
     3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc 
     3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc 
     3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc 
     4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa 
     4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat 
     4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa 
     4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt 
     4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa 
     4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac 
     4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg 
     4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt 
     4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac 
     4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag 
     4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac 
     4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat 
     4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg 
     4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg 
     4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta 
     4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca 
     4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg 
     5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg 
     5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc 
     5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt 
     5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg 
     5281 cagactaatg ggatcgagga aaacctgtac ttccaatcca attcgtggcg tgaactgatc 
     5341 gatggtcatg agaaactgac ccgtcgtcag tgccatcaac aagaggcggt gtgggaactg 
     5401 ctgcacaccg aggcgagcta catccgtaag ctgcgtgtga tcattaacct gttcctgtgc 
     5461 tgcctgctga acctgcagga aagcggcctg ctgtgcgagg ttgaagcgga gcgtctgttt 
     5521 agcaacatcc cggagattgc gcaactgcat cgtcgtctgt gggcgagcgt gatggcgccg 
     5581 gttctggaaa aagcgcgtcg tacccgtgcg ctgctgcagc cgggtgactt cctgaagggc 
     5641 ttcaaaatgt ttggtagcct gtttaaaccg tacatccgtt attgcatgga ggaagagggc 
     5701 tgcatggagt acatgcgtgg tctgctgcgt gacaacgacc tgttccgtgc gtatattacc 
     5761 tgggcggaaa agcacccgca gtgccaacgt ctgaaactga gcgatatgct ggcgaagccg 
     5821 caccaacgtc tgaccaaata tccgctgctg ctgaaaagcg tgctgcgtaa gaccgaagag 
     5881 ccgcgtgcga aagaagcggt ggttgcgatg atcggcagcg tggagcgttt cattcaccac 
     5941 gttaacgcgt gcatgcgtca gcgtcaagaa cgtcagcgtc tggcggcggt ggttagccgt 
     6001 atcgacgcgt acgaagtggt tgagagcagc agcgacgaag ttgataagct gctgaaagag 
     6061 tttctgcacc tggatctgac cgcgccgatt ccgggtgcga gcccggaaga aacccgtcaa 
     6121 ctgctgctgg aaggcagcct gcgtatgaaa gagggtaaag acagcaagat ggatgtgtat 
     6181 tgcttcctgt ttaccgacct gctgctggtg accaaggcgg ttaagaaagc ggaacgtacc 
     6241 cgtgttatcc gtccgccgct gctggttgac aaaattgttt gccgtgaact gcgtgatccg 
     6301 ggcagcttcc tgctgatcta cctgaacgag tttcacagcg cggtgggtgc gtataccttt 
     6361 caggcgagcg gtcaagcgct gtgccgtggt tgggttgaca ccatttacaa cgcgcagaac 
     6421 caactgcaac aactgcgtgc gcaggaaccg ccgggtagcc agcaaccgct gcagagcctg 
     6481 gaagaggaag aggatgagca agaagaggaa gaggaagagg aagaagagga agaagaaggc 
     6541 gaagacagcg gcaccagcgc ggcgagcagc ccgacctaat aattggaagt ggataacgga 
     6601 tccgcgatcg cggcgcgcca cctggtggcc ggccggtacc acgcgtgcgc gctgatccgg 
     6661 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 
     6721 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 
     6781 tatccggata tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca 
     6841 agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg 
     6901 ggtgcgcata gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg 
     6961 atgctgtcgg aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct 
     7021 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc 
     7081 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta 
     7141 tcgatgataa gctgtcaaac atgagaa 
//
