Question

Please solve Q4:

Q1) This DNA sequence v is for an actual plasmid named pSB1A2-113521. It contains the 5' to 3' DNA sequence for constitutive production of a red fluorescent protein in bacteria, along with a ColE1 origin of replication and a resistance gene for the antibiotic ampicillin. The DNA is circular, so if one ran along the DNA and hit the end of the sequence, one would find themselves back at the beginning. The sequence of the complementary strand is not shown.) If you were to cut this plasmid with the restriction enzymes Spel and Vspl, fill in the answer with the entire sequence linked to above, adding a hat "A" to indicate where the DNA would be cut. Your answer should consist only of lower-case nucleotides and hat “symbols. DNA Sequence: gaattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaag aggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttogtatggaaggttccgttaacggtcacg agttegaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctg ccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcaaacgttaaacacccggctgacatcccggactacctga aactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgca agacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgg gaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtca ctacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggaca tcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgata gtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgt cggtgaacgctctctactagagtcacactggctcaccttcggggggcctttctgcgtttatatactagtagcggccgctgcagttgcagg cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca gaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctgg cgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataa agataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttc gggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtcgctccaagctgggctgtgtgcacgaacc ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagca gccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactaga aggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgc tggtagcggtggttttttttttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctg acgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatg aagttttaaatcaattaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct atttogttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgata ccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgca actttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattg ctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccat gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagca ctgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggc gaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttct tcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttta ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaat actcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaaca aataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggc gtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctg

Answer 1

The questions are answered below. The questions are little vague, I have done my best. Feel free to get back in comments for clarifications.

1. The restriction site of Spel is 5' a^ctagt 3'
                                         3' tgatc^a 5'

The restriction site of VspI is 5' at^taat 3'
                                         3' taat^ta 5'

2. The ligation can occur between the restricted regions with SpeI and VspI. Each side cut by each enzyme. We will just fill the restriction site of Spe1 on left and VspI on the right side.
5'...actagt----MYDNA----attaat 3'
3'...tgatca----MYDNA----taatta 5'

1 3 7 10 11 12 13 14 Gaattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactacta gaga aagaggagaaat actagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttogtatggaaggttccgttaacggtcacgagttegaa atcgaaggtg aaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtegtccgctgccgttcgcttgggacatcctgtccccgcag ttccagtacggttccaaagcaaacgttaaacacccgectgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgegaacgtgttatg aacttcgaagacggtegtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtegtaccaacttcccgt ccgacggtccggttatgcagaaaaaaaccatgggttegga agcttccaccgaacgtatgtacccggaagacggtgctctga aaggtgaaatcaaaat gcgtctgaaactgaaagacggtegtcactacgacgctgaagttaa aaccacctacategctaaaaaaccggttcagctgccgggtgcttacaaaacc gacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacg ctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaa agactgegcctttogttttatctgttgtttgtcgg tgaacgctctctactagagtcacactggctcaccttcgggteggcctttctgcgtttatata"ctagtag ggccgctgcagttgcaggcttcctcgctca ctgactcgctgcgctcggtcgtteggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcagga aagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaa ggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggteg gaaacccgacaggactataaagataccaggcgtttccccctegaagctccctcgtgcgctctcctgtt ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagaca cgacttatcgccactegcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacg gctacactagaaggacagtatttogtatctgcgctctgctgaagccagttaccttcggaaaa agagttegtagctcttgatccrgcaaacaaaccacc gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga aaaa aaggatctcaagaagatcctttgatcttttctacggggtctgacgctc agtegaacgaaaactcacgttaagegattttggtcatg agattatcaaa aaggatcttcacctagatccttttaaattaaaa atgaagttttaa atcaa tetaaagtatatatgagta aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttogttcatccatagttgcct gactccccgtcgtgtagataactacgatacgegagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagat ttatcagcaataaaccagccagccggaagegccgagcgcagaagtegtcctgcaactttatccgcctccatccagtctatataattgttgccgegaag ctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagct ccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtteg ccgcagtgttatcactcategttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg actggtgagtactcaaccaagt cattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaa aagtgctcatca ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttegatgtaacccactcgtgcacccaactgatcttcagcatc ttttactttcaccagcgtttctgggtgagcaaaaacagga aggcaaa atgccgcaaaa aagggaataagggcgacacggaaatgttga atactcata ctcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata tttgaatgtatttagaaa aataaacaaataggggttccg cgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcag ataaaaaaaatccttagctttcgctaaggatgatttctg 17 18 21 22 24 26 27 28 29 30 31

3. The restriction enzumes cut the plasmid and obtain 2 fragments. The ligation would place the MYDNA between the cut sites of enzymes.

1 2 5 6 10 Gaattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaagaggagaaat actagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttogtatggaaggttccgttaacggtcacgagttegaaatcgaaggtg aaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcag ttccagtacggttccaaagcaaacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatg aacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgt ccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaat gcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaacc gacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacg ctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcgg tgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatata"ctagtMYDNAatataattgttgccgggaagctaga gtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggt tcccaacgatca aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca gtgttatcactcatggttatgg cagcactgcataattctcttactgtcatgccatccgta agatgcttttctgtgactggtgagtactcaaccaagtcatto tgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattgg aaaacgttcttcgggggaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatacto ttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcg cacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagat aaaaaaaatccttagctttcgctaaggatgatttctg 12 13 14 15 16 17 18 19

4. Tube 2 contains only DNA. It is resticted with enzymes, so it will have sticky ends like below.
5' ctagt---MYDNA--ta 3'