Restriction Free Cloning

Construct Name
pGW1-Insert

Forward Primer
Plasmid annealing = 62°C     Target annealing = 57°C    Length = 53
GGAGCAAAAGCTGATATCTGAAGAGGACTTGGAGAACGAAATCTTCACTCCCC


Reverse Primer
Plasmid annealing = 58°C     Target annealing = 56°C    Length = 55
CAATAAACAAGTTAACAACAACAATTGCATTCATTTTAGGAGCTTTGTTGCTCCC

1° PCR Size New Plasmid Size Insert Sites Insert Size
5601bps 10555bps
1839-1965
5535bps

2° PCR conditions
Extension Time ng of insert ng of plasmid
3:28 mins 1344.2 61.8

New construct
ATGC = Original plasmid sequence. ATGC = Inserted sequence.

ATCGCAGTTT CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA GAGGCGACAT CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG AATCAATATT GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT CAATATTGGC TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT ACATTTATAT TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGC GGCCGGGAAC GGTGCATTGG AACGCGGATT CCCCGTGCCA AGAGTGACGT AAGTACCGCC TATAGAGTCT ATAGGCCCAC CCCCTTGGCT TCTTATGCAT GCTATACTGT TTTTGGCTTG GGGTCTATAC ACCCCCGCTT CCTCATGTTA TAGGTGATGG TATAGCTTAG CCTATAGGTG TGGGTTATTG ACCATTATTG ACCACTCCCC TATTGGTGAC GATACTTTCC ATTACTAATC CATAACATGG CTCTTTGCAC AACTCTCTTT ATTGGCTATA TGCCAATACA CTGTCCTTCA GAGACTGACA CGGACTCTGT ATTTTTACAG GATGGGGTCT CATTTATTAT TTACAAATTC ACATATACAA CACCACCGTC CCCAGTGCCC GCAGTTTTTA TTAAACATAA CGTGGGATCT CCAGCGAATC TCGGGTACGT GTTCCGGACA TGGGGCTCTT CTCCGGTAGC GGCGGAGCTT CTACATCCAG CCCTGCTCCC ATCCTCCCAC TCATGGTCCT CGGCAGCTCC TTGCTCCTAA CAGTGGAGGC CAGACTTAGG CACAGCACGA TGCCCACCAC CACCAGTGTG CCCACAAGGC CGTGGCGGTA GGGTATGTGT CTGAAAATGA GCTCGGGGAG CGGGCTTGCA CCGCTGACGC ATTTGGAAGA CTTAAGGCAG CGGCAGAAGA AGATGCAGGC AGCTGAGTTG TTGTGTTCTG ATAAGAGTCA GAGGTAACTC CCGTTGCGGT GCTGTTAACG GTGGAGGGCA GTGTAGTCTG AGCAGTACTC GTTGCTGCCG CGCGCGCCAC CAGACATAAT AGCTGACAGA CTAACAGACT GTTCCTTTCC ATGGGTCTTT TCTGCAGTCA CCGTCCTTGA CACGAAGCTT GCCACCATGG AGCAAAAGCT GATATCTGAA GAGGACTTGG AGAACGAAAT CTTCACTCCC CTTCTGGAGC AGTTCATGAC CAGCCCCTTG GTCACTTGGG TTAAAACGTT TGGACCTCTG GCTGCAGGAA ATGGGACTAA CCTGGATGAG TATGTGGCTT TAGTGGATGG GGTATTCTTG AACCAGGTCA TGCTCCAAAT TAATCCTAAG TCGGAGAGTC AACGAGTAAA TAAAAAAGTC AACAATGATG CTTCCCTTAG AATTCACAAT CTGTCCATTT TGGTGAAACA GATAAAATTT TATTATCAGG AGACTTTGCA GCAGTTGATT ATGATGCCAT TGCCAGATAT CTTAATCATT GGCAAAAATC CCTTTTCTGA ACAAGGTACA GAAGAAGTTA AAAAGTTGCT TTTGCTCTTA CTTGGTTGTG CAGTTCAGTG TCAGAAAAAG GAAGAATTTA TTGAAAAAAT TCAAGGTTTA GATTTTGATA CGAAAGCAGC AGTTGCTGCA CACATTCAAG AGGTAACTCA TAATCAAGAA AATGTATTTG ATCTGCAATG GATGGAAGTG ACAGATATGT CTCAGGAAGA CATAGAACCA CTCTTAAAGA ATATGGTGTC ACATCTAAGA AGACTGATAG ATGAGAGAGA TGAACATTCA GAGACTATTG TAGAACTCTC AGAAGAGCGA GATGGTGTTC ATTTCCTACC CCATGCCTCT TCATCTGCAC AGTCACCCTG TGGTTCTCCA GGCATGAAGC GAACAGAAAG TCGACAGCAT CTCTCAGTGG AATTGGCTGA TGCTAAAGCC AAGATCCGAA GACTTAGGCA GGAATTGGAA GAAAAGACTG AGCAGTTACT GGACTGTAAA CAAGAACTTG AGCAAATAGA AGTGGAATTG AAGAGGCTGC AACAAGAGAA CATGAATTTG CTTTCGGATG CTCGATCTGC TAGAATGTAC CGAGATGAAT TGGATGCCCT CCGAGAGAAA GCAGTCAGAG TTGATAAGCT TGAAAGTGAA CTCAGTAGAT ATAAAGAGAG ACTACATGAT ATTGAATTTT ATAAGGCAAG AGTTGAGGAA TTAAAAGAAG ATAATCAAGT TTTATTAGAA ACAAAAACTA TGCTGGAAGA CCAACTAGAA GGAACTCGGG CTCGTTCTGA CAAACTACAT GAATTAGAAA AAGAGAATTT ACAACTAAAG GCTAAATTGC ATGATATGGA GATGGAACGA GATATGGATA GAAAGAAGAT TGAAGAATTA ATGGAAGAAA ATATGACTCT GGAAATGGCA CAGAAACAAA GTATGGATGA GTCATTACAC CTTGGCTGGG AGCTGGAACA GATATCCAGA ACTAGTGAAC TTGCTGAAGC ACCACAGAAG TCCTTGGGCC ATGAGGTAAA TGAATTAACA TCAAGTAAGT TATTAAAGTT GGAAATGGAA AACCAGAGTC TGACGAAAAC CGTAGAAGAG CTCCGGAGCA CTGCGGACTC CGCAGCAGGC AGCACTTCCA AAATCCTAAA AGTTGAAAAG GAAAACCAGA GACTGAATAA AAAGGTTGAA ATTCTTGAAA ATGAAATTAT TCAAGAAAAG CAAAGTCTTC AGAATTGTCA AAATTTAAGC AAGGATCTAA TGAAAGAGAA AGCTCAGCTT GAAAAAACAA TAGAGACGCT CAGAGAGAAT TCAGAGAGGC AGATTAAAAT ATTGGAGCAG GAAAATGAAC ATCTGAATCA AACAGTGTCT TCCTTACGGC AGCGCTCCCA GATTAGTGCT GAGGCGAGAG TGAAAGACAT TGAAAAAGAA AATAAAATTC TCCATGAATC TATCAAAGAA ACATGTGGAA AACTAAGCAA GATTGAATTT GAAAAAAGAC AAATGAAAAA GGAATTAGAG CTTTATAAAG AGAAAGGAGA GCGAGCAGAA GAACTTGAAA ACGAGTTGAA TCATCTTGGA AAGGAGAATG AATTGTTGCA GAAAAAGATA ACGAATTTAA AGATTACGTG TGAAAAACTT GAGACATTAG AACAGGAAAA TTCAGAGCTA GAAAGAGAAA ATAGGAAATT TAAAAAAACT TTGGACAGCT TTAAAAACCT TACTTTTCAG TTAGAATCTC TAGAAAAAGA GAATTCCCAA CTCGATGAAG AAAACTTAGA ACTGCGAAGG AGCGTGGAGT CTCTGAAGTG TGCGAGCATG AGAATGGCCC AGCTCCAGCT AGAGAACAAG GAGCTGGAGA GTGAGAAGGA GCAGCTGAGG AAGGGCTTGG AGCTCATGAG AGCCTCTTTC AAGAAGACAG AACGCTTGGA AGTTAGCTAC CAGGGGCTAG ACACAGAAAA TCAAAGACTA CAAAAAGCTC TAGAAAATAG CAATAAAAAA ATCCAACAGT TAGAGAGTGA GCTACAAGAC TTAGAGATGG AAAATCAAAC ATTACAAAAA AGCCTAGAGG AACTGAAAAT ATCTAGCAAA AGACTGGAAC AACTAGAAAA AGAAAACAAG TCATTAGAGC AAGAGACATC TCAACTTGAA AAGGACAAAA AACAATTGGA GAAGGAAAAT AAACGACTCC GACAACAAGC AGAAATAAAA GATACCACAT TAGAAGAAAA TAATGTGAAA ATTGGGAATT TGGAAAAAGA AAACAAAACT TTATTCAAAG AAATTAATGT ATATAAAGAG TCCTGTGTTC GTCTAAAAGA ATTAGAAAAA GAAAATAAAG AGCTTGTAAA AAGAGCAACT ATTGATATTA AAACCTTGGT TACATTACGT GAGGATTTAG TCAGTGAGAA ATTGAAGACA CAACAAATGA ACAATGATCT TGAGAAATTA ACTCATGAGC TTGAGAAGAT AGGTTTAAAT AAAGAGCGGC TCCTACACGA TGAGCAGAGC ACTGATGACA GTAGGTACAA ACTTTTGGAA TCAAAATTAG AATCCACTCT TAAGAAGTCC CTTGAAATAA AAGAAGAAAA AATTGCTGCT TTAGAAGCTC GGTTAGAAGA ATCTACAAAT TACAATCAGC AATTGCGCCA CGAACTTAAA ACAGTGAAAA AAAATTATGA AGCTCTCAAA CAGAGACAAG ATGAAGAAAG GATGGTACAG AGTTCCATTC CAGTCTCTGG AGAAGATGAC AAGTGGGGCC GAGAAAGTCA AGAAGCGACT AGAGAGCTCC TGAAAGTTAA AGACAGGTTA ATTGAAGTAG AAAGAAACAA TGCTACGCTG CAAGCAGAGA AGCAAGCACT GAAAACTCAA CTGAAGCAAC TTGAAACACA AAATAATAAT TTGCAGGCTC AGATTCTTGC ACTGCAGAGG CAGACCGTGT CATTGCAGGA GCAGAATACC ACGCTTCAGA CACAGAATGC CAAGCTTCAG GTTGAAAATT CTACCCTTAA TTCACAAAGT ACTTCACTTA TGAACCAGAA TGCACAGCTT CTAATCCAGC AGTCTTCCTT AGAAAATGAA AATGAATCTA TAATGAAAGA ACGAGAAGAC TTGAAATCTC TGTATGATGC TCTGATAAAA GATCACGAAA AGCTGGAACT TCTTCATGAG CGGCAAGCTT CAGAGTATGA GTCTCTGATC TCTAAACATG GCACCCTGAA GTCTGCTCAC AAAAATCTTG AAGTGGAACA TAAAGACCTT GAAGACCGTT ACAATCAGTT ATTAAAACAG AAAGGACAAT TGGAAGATTT GGAAAAAATG ATCAAAACGG AACAGGAAAA AATGCTGCTT GAAAGCAAAA ACCATGAAGT GGTAGCTTCA GAGTACAAGA AGCTTTGTGG TGAAAATGAT AGGTTAAATT ATACATATAG TCAGCTTTTA AAAGAGACTG AAATTTTACA AATGGATCAT AAAAATTTGA AAAGTGTTCT AAATAATTCT AAATTGGAAC AAACAAGATT AGAAGCTGAA TTTTCAAAGC TAAAGGAGCA GTATCAGCAA CTAGATATCA CATCTACCAA GCTAAATAAC CAGTGTGAGT TGCTCAGCCA ACTTAAAGGA AATTTAGAAG AAGAAAATCG ACATCTACTT GATCAGATCC AGACACTAAT GCTACAGAAC AGAACACTGC TGGAGCAAAA TATGGAAAGC AAGGATCTCT TTCATGTTGA ACAAAGACAG TACATTGATA AATTAAATGA ATTGAGACGA CAAAAAGAAA AATTAGAAGA GAAAATTATG GATCAATACA AATTTTATGA CCCATCTCCT CCTAGAAGGA GAGGCAACTG GATTACTCTA AAAATGAGAA AATTGATAAA GTCAAAAAAA GATATTAATC GGGAACGTCA GAAATCTCTA ACACTAACAC CCACCCGCTC AGACTCCAGT GAAGGATTTC TTCAGCTGCC CCATCAAGAC AGCCAAGACA GCTCTTCAGT AGGATCAAAC TCTTTAGAAG ATGGCCAAAC CCTGGGGACC AAGAAGAGCA GCACCATGAA TGACCTGGTG CAGTCCATGG TCCTAGCAGG AGGACAGTGG ACAGGTAGTA CTGAAAATTT GGAGGTCCCT GATGATATTT CAACGGGTAA AAGGAGAAAA GAATTGGGAG CTATGGCCTT CTCTACTACA GCCATCAACT TTTCAACTGT CAACTCTTCT GCAGCCTTCC GATCCAAGCA GTTGGTTAAT AATAAAGATA CTACATCCTT TGAAGACATA AGTCCACAAG GTATTAGTGA TGATTCTAGT ACGGGATCAA GAGTTCATGC TTCAAGACCA GCCAGCCTTG ATAGTGGCAG AACATCCACT AGCAATAGCA ATAATAATGC TTCACTCCAT GAAGTCAAAG CAGGTGCAGT TAATATCCAA AGCAGGCCAC AAAGCCACAG CAGTGGAGAT TTCAGTCTGC TTCATGATCA TGAGACTTGG TCTAGCAGTG GTAGCAGTCC AATTCAGTAT TTGAAAAGAC AGACCAGGTC AAGCCCTATG CTCCAGCACA AAATATCTGA AACAATAGAG AGTCGAGCCC ATCACAAGAT GAAAGCTGGC TCCCCAGGAA GTGAAGTTGT TACTCTACAA CAGTTTTTGG AAGAAAGCAA CAAGCTTACC TCAATACAGT TGAAGTCCTC GAGTCAAGAG AATCTTTTAG ATGAAGTAAT GAAAAGTTTG TCTGTGTCTT CCGACTTTCT GGGGAAAGAC AAGCCAGTTA GCTGTACCCT GGCCAGGTCA GTAAGTGGAA AGACTCCAGG GGACTTCTAT GATAGACGGA CAACTAAGCC TGAATTTTTG AGAACTGGTC CTCAAAAGAC TGAAGATGCC TACACCATCA GCTCTGCAGG GAAACCTACA CCAAGCACTC AAGGAAAGAT AAAACTAGTA AAAGAAACCT CTGTGTCAAG ACAATCAAAA GATAGCAATC CTTATGCCAC TTTACCTCGT GCAAGCAGTG TGATCTCTAC TGCTGAAGGA ACAACTCGAA GGACGAGCAT CCATGATTTT TTGAGCAAAG ACAGTAGACT GCCCGTGTCA GTCGATTCAT CACCACCTAC TGCTGGTAGC AGCAGCACTA CAGCATCTAA TGTGAACAAA GTACAAGAGA GCAGAAATTC AAAAAGCAGG TCTAGGGAGC AACAAAGCTC CTAAAATGAA TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTATC ATGTCTGGAT CCGTACCGAG CTCGCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATT GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATAAGCGGC CGCGGCCATG CCGGCCACTA GTCTCGAGTT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GTCTCGCGCG TTTCGGTGAT GACGGTGAAA ACCTCTGACA CATGCAGCTC CCGGAGACGG TCACAGCTTG TCTGTAAGCG GATGCCGGGA GCAGACAAGC CCGTCAGGGC GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT GAAGACGTCG CCTCCTCACT ACTTCTGGAA TAGCTCAGAG GCCGAGGCGG CCTCGGCCTC TGCATAAATA AAAAAAATTA GTCAGCGCGC CATTCGCCAT TCAGGCTGCG CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TGGCGAAAGG GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CACGACGTTG TAAAACGACG GCCAGTGCCA TCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG CAAAT

Apr 19 2024



Plasmid markers and restriction enzymes:







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