Part:BBa_K2993003

Fusion protein of ARRAY2-GSAT-C-TEV-6xHisTag

This part contains the C-TEV split protein fused to zincfinger array 2 via a GSAT-linker.

This fusion protein should be used with its counterpart which contains the C-TEV protein fused to another zinc-finger array with a binding affinity for a different DNA sequence. On our iGEMteam_Rotterdam2019 webpage, you can read all about how to use this part properly.

This part is special in its own way because it actually is a fusion of one half of a protein to a whole array of zinc-finger proteins. Much like a multi fusion protein.

The full sequence of this part is (we tried fitting it in the page width):

GAATTCGCGGCCGCTTCTAGAGGTTGACGGCTAGCTCAGTCCTAGGTACAG TGCTAGCTtcaagaGAAAGAGGGGACAAaCTAGATGGCCGGCCTGGAACCAGGCGAAAAA CCGTACAAATGCCCGGAATGTGGTAAAAGCTTCAGCCAGAGCGGCCATCTG ACCGAACACCAGCGTACGCATACCGGCGAAAAACCATATAAATGTCCGGAATGTGGCAAA TCTTTTAGCCGTGAAGATAACCTTCACACCCATCAGCGCACCCATACTGGTG AAAAACCGTATAAGTGCCCGGAATGTGGCAAAAGTTTTAGCAGCAAGAAACATCTGGCG GAACATCAACGCACCCACACCGGCGAAAAGCCGTATAAATGCCCGGAATGCG GCAAAAGCTTTAGCCGCACCGATACCCTGCGCGATCACCAACGCACTCACACAGGTGA GAAACCCTACAAGTGCCCAGAATGTGGAAAATCATTTAGCCGCGAGGATAAT TTACACACCCATCAACGTACGCATACAGGCGAAAAGCCATACAAGTGTCCCGAGTGTGG CAAAAGTTTCTCGCGCGAAGATAATTTGCATACGCACCAACGCACGCATACT GGCAAGAAGACAAGTACCGGCGGAGGGTCGGCAGGAGGGAGTGGTTCCGGATCTAGTG GTGGAAGCTCAGGAGCTAGTGGTACAGGCACTGCGGGTGGCACTGGCAGCG GTTCCGGTACTGGCTCTGGCACCGGCaagagcatgtctagcatggtgtcagacactagctgcacatt cccttcatctgatggcatattctggaagcattggattcaaaccaaggatgggcagtgtggcagtccat tagtatcaactagagatgggttcattgttggtatacactcagcatcgaatttcaccaacacaaacaattatttcacaa gcgtgccgaaaaacttcatggaattgttgacaaatcaggaggcgcagcagtgggttagtggttgg cgattaaatgctgactcagtattgtgggggggccataaagttttcatggtgaaacctgaagagccttttcagccagtt aaggaagcgactcaactcatgaatACCGGCCACCACCATCACCATCACCATCACTACTAGTAGCGGCCGCTGCAG

Even though the protein has not been brought to expression, theoretical estimations were made with known software from ExPASy (https://web.expasy.org/protparam/) as shown below:

ProtParam User-provided sequence:

       10         20         30         40         50         60 

MAGLEPGEKP YKCPECGKSF SQSGHLTEHQ RTHTGEKPYK CPECGKSFSR EDNLHTHQRT

       70         80         90        100        110        120 

HTGEKPYKCP ECGKSFSSKK HLAEHQRTHT GEKPYKCPEC GKSFSRTDTL RDHQRTHTGE

      130        140        150        160        170        180 

KPYKCPECGK SFSREDNLHT HQRTHTGEKP YKCPECGKSF SREDNLHTHQ RTHTGKKTST

      190        200        210        220        230        240 

GGGSAGGSGS GSSGGSSGAS GTGTAGGTGS GSGTGSGTGK SMSSMVSDTS CTFPSSDGIF

      250        260        270        280        290        300 

WKHWIQTKDG QCGSPLVSTR DGFIVGIHSA SNFTNTNNYF TSVPKNFMEL LTNQEAQQWV

      310        320        330        340 

SGWRLNADSV LWGGHKVFMV KPEEPFQPVK EATQLMNTGH HHHHHHHY

References and documentation are available.

Number of amino acids: 348

Molecular weight: 38135.93

Theoretical pI: 8.83

Amino acid composition: Ala (A) 9 2.6% Arg (R) 13 3.7% Asn (N) 11 3.2% Asp (D) 10 2.9% Cys (C) 14 4.0% Gln (Q) 14 4.0% Glu (E) 23 6.6% Gly (G) 44 12.6% His (H) 28 8.0% Ile (I) 4 1.1% Leu (L) 13 3.7% Lys (K) 29 8.3% Met (M) 6 1.7% Phe (F) 14 4.0% Pro (P) 19 5.5% Ser (S) 40 11.5% Thr (T) 35 10.1% Trp (W) 5 1.4% Tyr (Y) 8 2.3% Val (V) 9 2.6% Pyl (O) 0 0.0% Sec (U) 0 0.0%

(B)   0	  0.0%
(Z)   0	  0.0%
(X)   0	  0.0%

Total number of negatively charged residues (Asp + Glu): 33 Total number of positively charged residues (Arg + Lys): 42

Atomic composition:

Carbon C 1631 Hydrogen H 2486 Nitrogen N 502 Oxygen O 523 Sulfur S 20

Formula: C1631H2486N502O523S20 Total number of atoms: 5162

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 40295 Abs 0.1% (=1 g/l) 1.057, assuming all pairs of Cys residues form cystines

Ext. coefficient 39420 Abs 0.1% (=1 g/l) 1.034, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).

                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 33.56 This classifies the protein as stable.

Aliphatic index: 29.14

Grand average of hydropathicity (GRAVY): -1.082

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 672
  Illegal SapI.rc site found at 982