Part:BBa_K2993000

Fusion protein of N-TEV-GSAT-ZincFingerArray1

This part contains the N-TEV split protein fused to a zincfinger array 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 composite is as follows: GAATTCGCGGCCGCTTCTAGAGCCTGCAATGACGGACGTATTCCTCTGGCCTCAACGCAAGATTGGCAGCTGAAACCGCCTTTCCAAAGTGA GTCCTTCGTCTATACGTTGCTTCTCAACGTGGCAAGTGACAGCACCACTGTGAACCAAACGTCCCGTTTCCACGTACCCAAGGGTGCCCAAT CTTGTATTAAGCattaaagaggagaaaaCTAGATGGCCGGCCACCACCATCACCATCACCATCACACCGGCggagaaagcttgtttaagggg ccgcgtgattacaacccgatatcgagcaccatttgtcatttgacgaatgaatctgatgggcacacaacatcgttgtatggtattggatttgg tcccttcatcattacaaacaagcacttgtttagaagaaataatggaacactgttggtccaatcactacatggtgtattcaaggtcaagaaca ccacgactttgcaacaacacctcattgatgggagggacatgataattattcgcatgcctaaggatttcccaccatttcctcaaaagctgaaa tttagagagccacaaagggaagagcgcatatgtcttgtgacaaccaacttccaaactACCGGCGGAGGGTCGGCAGGAGGGAGTGGTTCCGG ATCTAGTGGTGGAAGCTCAGGAGCTAGTGGTACAGGCACTGCGGGTGGCACTGGCAGCGGTTCCGGTACTGGCTCTGGCACCGGCCTGGAGC CGGGTGAAAAACCGTACAAATGCCCTGAATGCGGTAAAAGCTTTAGTCGCAGCGACAAACTTGTGCGCCACCAGCGTACTCACACCGGCGAA AAACCATATAAATGCCCGGAGTGTGGCAAAAGCTTCAGTCGCGAAGATAACCTTCACACCCACCAGCGCACCCATACCGGCGAAAAGCCGTA TAAATGCCCAGAATGTGGCAAAAGCTTTAGCCATCGCACGACCCTGACCAACCACCAGCGCACCCACACCGGCGAGAAACCGTATAAATGTC CCGAGTGTGGAAAGTCTTTCTCTACAAGTGGCCATTTGGTGCGTCATCAACGTACTCACACCGGAGAAAAGCCGTACAAATGCCCAGAATGC GGTAAGTCCTTCTCTCGTGAGGACAACTTGCATACCCATCAACGCACGCACACGGGTGAGAAACCATATAAGTGCCCCGAGTGCGGAAAATC GTTTTCTCAGTCAAGTTCCTTAGTCCGCCATCAGCGCACACACACTGGGAAGAAGACTAGCTACTAGTAGCGGCCGCTGCAG

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 

MAGHHHHHHH HTGGESLFKG PRDYNPISST ICHLTNESDG HTTSLYGIGF GPFIITNKHL

       70         80         90        100        110        120 

FRRNNGTLLV QSLHGVFKVK NTTTLQQHLI DGRDMIIIRM PKDFPPFPQK LKFREPQREE

      130        140        150        160        170        180 

RICLVTTNFQ TTGGGSAGGS GSGSSGGSSG ASGTGTAGGT GSGSGTGSGT GLEPGEKPYK

      190        200        210        220        230        240 

CPECGKSFSR SDKLVRHQRT HTGEKPYKCP ECGKSFSRED NLHTHQRTHT GEKPYKCPEC

      250        260        270        280        290        300 

GKSFSHRTTL TNHQRTHTGE KPYKCPECGK SFSTSGHLVR HQRTHTGEKP YKCPECGKSF

      310        320        330        340 

SREDNLHTHQ RTHTGEKPYK CPECGKSFSQ SSSLVRHQRT HTGKKTSY

Number of amino acids: 348

Molecular weight: 38330.65

Theoretical pI: 9.52

Amino acid composition: Ala (A) 4 1.1% Arg (R) 21 6.0% Asn (N) 10 2.9% Asp (D) 8 2.3% Cys (C) 14 4.0% Gln (Q) 13 3.7% Glu (E) 20 5.7% Gly (G) 45 12.9% His (H) 29 8.3% Ile (I) 10 2.9% Leu (L) 18 5.2% Lys (K) 28 8.0% Met (M) 3 0.9% Phe (F) 15 4.3% Pro (P) 21 6.0% Ser (S) 35 10.1% Thr (T) 38 10.9% Trp (W) 0 0.0% Tyr (Y) 9 2.6% Val (V) 7 2.0% 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): 28 Total number of positively charged residues (Arg + Lys): 49

Atomic composition:

Carbon C 1645 Hydrogen H 2564 Nitrogen N 520 Oxygen O 510 Sulfur S 17

Formula: C1645H2564N520O510S17 Total number of atoms: 5256

Extinction coefficients:

This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient.

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

Ext. coefficient 14285 Abs 0.1% (=1 g/l) 0.373, assuming all pairs of Cys residues form cystines

Ext. coefficient 13410 Abs 0.1% (=1 g/l) 0.350, 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 33.71 This classifies the protein as stable.

Aliphatic index: 38.36

Grand average of hydropathicity (GRAVY): -1.035

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 339