EcoFlex – User Guide
To provide the user with details for customizable modification of plasmids, we provide details of cloning procedures used.
ORFs
ORFs can be amplified by PCR from plasmid or genomic DNA, or synthesised to remove rare codons and excluded restriction sites (NdeI, BamHI, BsaI, BsmBI). Alternatively, genes with internal NdeI and BamHI sites can be PCR amplified with primers containing flanking BsaI sites or sub-cloned using an optional NcoI (CCATGG) site located in pBP-ORF, which also provides a start codon. Additionally, if BamHI is present within a gene, the 3’ primer can incorporate a BglII site, which forms cohesive overhangs with BamHI.
Bioparts
Users can customize the library with alternative DNA parts (promoters, RBS, tags etc) using the pBP-lacZa plasmid. Whilst we provide N-terminal His6 and Strep(II) tags; further fusion tags such as C-terminal extensions and fluorescence or probe tags can be custom built.
Plasmid purification and sequencing
Single colonies were grown overnight with antibiotic in 3-5 mL LB in a 50 mL Falcon. All plasmids were purified with a QIAprep Spin Miniprep kit and eluted with 50-100 μL of sterile (pre-heated 65°C for plasmids larger than 10 kb) distilled water to obtain ~60-200 ng/ul dsDNA. The yield varies depending on insert size, whilst decreased yields can be obtained with the promoter parts (SJM901 and SJM911) that exhibit toxicity from the GFP reporter.
Oligo annealing
Annealing primers were prepared at 20 mM in 25 m of 1 × T4 DNA ligase buffer (10 × NEB), heated to 90°C for 1 min, before cooling on ice. Annealed primers were phosphorylated with T4 Polynucleotide kinase for 1 hour at 37°C before dilution to 200 nM and ligation into the backbone plasmid. Standard ligations were prepared in 2 × Rapid Ligation buffer (Promega) and T4 DNA ligase (Promega) at room temperature for 30 min. Ligation mixtures were transformed into DH10b, JM109 or KRX chemically competent cells. For routine Level 1 transformations, DH10b competent cells were prepared with the calcium chloride method. For Level 2 transformations, commercial competent cells were used (competency 108 CFU/mg).
Golden Gate Reaction – Level 1
20 units of BsaI-HF (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and cycled 15 times with 5 min at 37 °C and 10 min 16 °C. This was followed by 5 min incubations at 50°C and 80°C.
Golden Gate Reaction – Level 2
10 units of BsmBI (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and incubated at 37°C for 16 hours. This was followed by a 5 min incubation at 80°C. Alternatively a 30 cycle protocol equivalent to Level 1 assembly can be used. This increases the total number of colonies obtained, but the percentage of correct clones decreases when using GFP as a positive marker for a 5 TU assembly.
Golden Gate Reaction – Level 3
20 units of BsaI-HF (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and cycled 30 times with 5 min at 37°C and 10 min 16°C. This was followed by 5 min incubations at 50°C and 80°C.
Secondary module cloning
A Level 2 unit containing 2, 3, 4 or 5 TUs can be sub-cloned with BsaI into two cohesive BpiI sites located in pTUS-A, -a and –b. The assembled plasmid is then ready for standard Level 2 assembly to accommodate up to 10 TUs.
EcoFlex kit plasmids – Assembly plasmids and “Destination Vectors”
Level 0
pBP-lacZ (#72948)
Promoters, RBS, Tags and terminators ligated between NdeI and SphI
ATCTAGAGACGCATATG-lacZa-GCATGCCGTCTCATTAG
pBP-ORF (#72949)
Open reading frames cloned between NdeI and BamHI
GGTCTCACATATGGGTTCCATGGGGCACGGATCCTCGAAGAGACC
Level 1 -[NM]- Negative marker rfp or lacZa fragment
pTU1-A (#72935 and #72939)
CGTCTCAATCTCTATAGAGACC-[NM]-GGTCTCATGTTTGCCAGAGACG
pTU1-B (#72936 and #72940)
CGTCTCATGCCCTATAGAGACC-[NM]-GGTCTCATGTTCCGGAGAGACG
pTU1-C (#72937 and #72941)
CGTCTCACCGGCTATAGAGACC-[NM]-GGTCTCATGTTGAAGAGAGACG
pTU1-D (#72938 and #72942)
CGTCTCAGAAGCTATAGAGACC-[NM]-GGTCTCATGTTTTAGAGAGACG
pTU1-D1 (#72943)
CGTCTCAGAAGCTATAGAGACC-[NM]-GGTCTCATGTTCTTCAGAGACG
pTU1-E (#72944)
CGTCTCACTTCCTATAGAGACC-[NM]-GGTCTCATGTTTTAGAGAGACG
Level 2
pTU2-A (#72950 and #72954)
GGTCTCACTATATCTAGAGACG-[NM]-CGTCTCATTAGGTACAGAGACC
pTU2-B (#72951 and #72955)
GGTCTCAGTACATCTAGAGACG-[NM]-CGTCTCATTAGGGACAGAGACC
pTU2-C (#72952 and #72956)
GGTCTCAGGACATCTAGAGACG-[NM]-CGTCTCATTAGTCGAAGAGACC
pTU2-D (#72953 and #72957)
GGTCTCATCGAATCTAGAGACG-[NM]-CGTCTCATTAGTGTTAGAGACC
pTU2-a (#72958)
GGTCTCACTATATCTAGAGACG-[RFP]-CGTCTCACCGGGTACAGAGACC
pTU2-b (#72959)
GGTCTCACTATATCTAGAGACG-[RFP]-CGTCTCAGAAGGTACAGAGACC
Level 3
pTU3-A – 2 fragments from Level 2 (#72945)
CGTCTCAATCTCTATAGAGACC-[RFP]-GGTCTCAGGACTTAGAGAGACG
pTU3-B – 4 fragments from Level 4 (#72946)
CGTCTCAATCTCTATAGAGACC-[RFP]-GGTCTCATGTTTTAGAGAGACG
Secondary module (Level 2) site – (pTU2S-a, -b and –A)
#73010 – pTU2S-a (pMB1 origin) Accepts 2 TUs
#73011 – pTU2S-b (pMB1 origin) Accepts 3 TUs
Placed between AatI and EcoRI sites in pTU2–A, pTU2-a and pTU2-b. Modules from pTU2-A are sub-cloned (by gel extraction) as a BsaI fragment into the compatible BpiI sites. NdeI and SphI are placed between the BpiI sites for further modification and spacing between the restriction sites.
CTATAAGTCTTCCATATGCACGCATGCGAAGACAAGTAC
Biological Parts
Promoters
J23100 (#72963)
CTATTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGTAC
J23108 (#72964)
CTATCTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTAC
J23114 (#72965)
CTATTTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCGTAC
SJM901 (#72966)
CTATTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTAC
SJM902 (#72967)
CTATTTTACAGCTAGCTCAGTCCTAGGGATTATGCTAGCGTAC
SJM903 (#72968)
CTATCTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGCGTAC
SJM905 (#72969)
CTATTTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGCGTAC
SJM906 (#72970)
CTATTTGATGGCTAGCTCAGTCCTAGGGATTGTGCTAGCGTAC
SJM908 (#72971)
CTATTTTATAGCTAGCTCAGCCCTTGGTATTATGCTAGCGTAC
SJM910 (#72972)
CTATTTGATGGCTAGCTCAGTCCTTGGTATTATGCTAGCGTAC
SJM911 (#72973)
CTATTTGACAGCTAGCTCAGTCCTTGGTACTGTGCTAGCGTAC
SJM912 (#72974)
CTATTTGATAGCTAGCTCAGTCCTAGGTACTATGCTAGCGTAC
SJM915 (#72976)
CTATTTTATGGCTAGCTCAGTCCTTGGTATTATGCTAGCGTAC
T7 consensus (#72977)
CTATTAATACGACTCACTATAGGGAGAGTAC
T7 promoter and PET RBS combinations
T7 promoter, lac operator and PET RBS (#72978)
CTATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATA
T7 promoter, lac operator, PET RBS, His6-tag and thrombin site (#72989)
CTATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATA
RBS, linkers and tags
BBa_B0034 (#72980)
GTACAAAGAGGAGAAACATA
PET RBS (#72981)
GTACTTTAACTTTAAGAAGGAGATATACATA
N-terminus tag-linker (#72982)
GTACTTTAACTTTAAGAAGGAGATATATAAA
Hexahistidine (#72983)
TAAATGCACCATCACCATCACCATA
Strep(II) (#72984)
TAAATGTGGAGCCACCCGCAGTTCGAAAAACCGCATA
TL1 (#72985)
GTACAGATCTAATAATTTTGTTTAACTTTGGGGGGATACATA
TL2 (#72986)
GTACAGATCTAATAATTTTGTTTAACTTTGGGAGGATACATA
TL3 (#72987)
GTACAGATCTAATAATTTTGTTTAACTTTGGGGGAATACATA
TL4 (#72988)
GTACAGATCTAATAATTTTGTTTAACTTTAAGGAGATACATA
TL5 (#72989)
GTACAGATCTAATAATTTTGTTTAACTTTGGAGAAATACATA
TL6 (#72990)
GTACAGATCTAATAATTTTGTTTAACTTTGGGGAAATACATA
TL7 (#72991)
GTACAGATCTAATAATTTTGTTTAACTTTAGGGGAATACATA
TL8 (#72992)
GTACAGATCTAATAATTTTGTTTAACTTTAAAGAGATACATA
TL9 (#72993)
GTACAGATCTAATAATTTTGTTTAACTTTAAAAAGATACATA
TL10 (#72994)
GTACAGATCTAATAATTTTGTTTAACTTTGAAAAAATACATA
TL11 (#72995)
GTACAGATCTAATAATTTTGTTTAACTTTAAAGGGATACATA
TL12 (#72996)
GTACAGATCTAATAATTTTGTTTAACTTTGGGAAGATACATA
Open reading frames
GFP (#72960)
CATATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGAGCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATCGA
CFP (#72961)
CATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCTAAGGATCCTCGA
mCherry (#72962)
CATATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGGATCCTCGA
iGEM terminators
BBa_B0012 (#72997)
TCGATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATGTT
BBa_B0015 (#72998)
TCGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATGTT
Synthetic terminators
L3S2P21 (#72999)
TCGACTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCTGTT
L3S1P51 (#73000)
TCGAAAAAAAAAAAAAGGCCTCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAATGTT
L3S1P32 (#73001) – BsmBI site underlined
TCGAGACGAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTATTTTTCAACAAAATGTT
L3S1P11 (#73002) – BsmBI site underlined
TCGAGACGAACAATAAGGCCTCCCTTCGGGGGGGCCTTTTTTATTGATAACAAAATGTT
L2U8H11 (#73003)
TCGATAGCGTGCTAACCACGCACGCTATTGTTGTATTGTT
L2U5H11 (#73004)
TCGATAGCGTGCGAACAGCACGCTATTGTTGTATTGTT
L2U5H08 (#73005)
TCGATGCCGGGAGAACATCCCGGCATTGTTGTATTGTT
L2U3H03 (#73006)
TCGATAGCGTGACCGGCGCATCGGTCACGCTATTTGTTGAGTGTT
L2U2H09 (#73007)
TCGAACGGCCCTCGCAAGGGCCGTTTTTTTGTATGTT
L1U1H09 (#73008)
TCGACGACGATGTTCGCATCGTCGTTTTTTTTTTGTT
L1U1H08 (#73009)
TCGACCCGCATGTTCGCATGCGGGTTTTTTTTTTGTT
Individual plasmids
Secondary module (Level 2) site – (pTU2S-a, -b and –A)
#74088 – pTU2S-a (p15A origin) Accepts 2 TUs
#74089 – pTU2S-b (p15A origin) Accepts 3 TUs
#74090 – pTU2S-A (p15A origin) Accepts 4-5 TUs
#74091 – pTU2S-a (colE1 origin) Accepts 2 TUs
#74092 – pTU2S-b (colE1 origin) Accepts 3 TUs
#74093 – pTU2S-A (colE1 origin) Accepts 4-5 TUs
Lactate promoter - Bba_K822000 (#74094)
CTATCACATTCCTATAGGCCGAGTAAGGTGTTCACGCCGCATCCGGCAAGATAAGGCGCTCTGGATCAACAACCTAAGGGCAATTCTCTGATGAGGATTGCCCTTTTCTTTACCAGACATCTCCCCCCACAAGAATTGGCCCTACCAATTCTTCGCTTATCTGACCTCTGGTTCACAATTTCCCAATTAAAACTCACATCAATGTTGCCAATACATAACATTTAGTTAACCATTCATTGTCATTATCCCTACACAACACAATTGGCAGTGCCACTTTTACACAACGTGTGACAAGGAGATGAGCAACAGACTCATTACACGATGTGCGTGGACTCCGTAC
lldR – E. coli MG1655 (#74095)
CATATGATTGTTTTACCCAGACGCCTGTCAGACGAGGTTGCCGATCGTGTGCGGGCGCTGATTGATGAAAAAAACCTGGAAGCGGGCATGAAGTTGCCCGCTGAGCGCCAACTGGCGATGCAACTCGGCGTATCACGTAATTCACTGCGCGAGGCGCTGGCAAAACTGGTGAGTGAAGGCGTGCTGCTCAGTCGACGCGGCGGCGGGACGTTTATTCGCTGGCGTCATGACACATGGTCGGAGCAAAACATCGTCCAGCCGCTAAAAACACTGATGGCCGATGATCCGGATTACAGTTTCGATATTCTGGAAGCCCGCTACGCCATTGAAGCCAGCACCGCATGGCATGCGGCAATGCGCGCCACACCTGGCGACAAAGAAAAGATTCAGCTTTGCTTTGAAGCAACGCTAAGTGAAGACCCGGATATCGCCTCACAAGCGGACGTTCGTTTTCATCTGGCGATTGCCGAAGCCTCACATAACATCGTGCTGCTGCAAACCATGCGCGGTTTCTTCGATGTCCTGCAATCCTCAGTGAAGCATAGCCGTCAGCGGATGTATCTGGTGCCACCGGTTTTTTCACAACTGACCGAACAACATCAGGCTGTCATTGACGCCATTTTTGCCGGTGATGCTGACGGGGCGCGTAAAGCAATGATGGCGCACCTTAGTTTTGTTCACACCACCATGAAACGATTCGATGAAGATCAGGCTCGCCACGCACGGATTACCCGCCTGCCCGGTGAGCATAATGAGCATTCGAGGGAGAAAAACGCATAAGGATCCTCGA
T7 RNA polymerase (#74096)
CATATGaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaTCGA