New plasmid
MoClo level 1 destination vector pL1F-1
from Bartlett lab
amp-r
Lac-Z cassette
Test for restriction sites:
per 20 uL reaction:
10 uL midiprep DNA (40 ng/uL)
2 uL 10x NEB cut smart
1 uL enzyme (all from NEB) or water
7 uL water
- water/control
- BamH1
- EcoR1
- Kpn
incubate 1 hr at 37
Mix with 4 uL loading dye
Run in 1% agarose gel ~1.5 hr (needs a long run - the DNA pieces are long)
See attached picture below
Sequence
1 gtggggccca ctgcatccac cccagtacat taaaaacgtc cgcaatgtgt tattaagttg
61 tctaagcgtc aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc
121 cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag tcagatcagg
181 atctcctttg cgacgctcac cgggctggtt gccctcgccg ctgggctggc ggccgtctat
241 ggccctgcaa acgcgccaga aacgccgtcg aagccgtgtg cgagacaccg cggccgccgg
301 cgttgtggat acctcgcgga aaacttggcc ctcactgaca gatgaggggc ggacgttgac
361 acttgagggg ccgactcacc cggcgcggcg ttgacagatg aggggcaggc tcgatttcgg
421 ccggcgacgt ggagctggcc agcctcgcaa atcggcgaaa acgcctgatt ttacgcgagt
481 ttcccacaga tgatgtggac aagcctgggg ataagtgccc tgcggtattg acacttgagg
541 ggcgcgacta ctgacagatg aggggcgcga tccttgacac ttgaggggca gagtgctgac
601 agatgagggg cgcacctatt gacatttgag gggctgtcca caggcagaaa atccagcatt
661 tgcaagggtt tccgcccgtt tttcggccac cgctaacctg tcttttaacc tgcttttaaa
721 ccaatattta taaaccttgt ttttaaccag ggctgcgccc tgtgcgcgtg accgcgcacg
781 ccgaaggggg gtgccccccc ttctcgaacc ctcccggccc gctaacgcgg gcctcccatc
841 cccccagggg ctgcgcccct cggccgcgaa cggcctcacc ccaaaaatgg cagcgctggc
901 caattcgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
961 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
1021 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
1081 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
1141 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
1201 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
1261 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
1321 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
1381 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
1441 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
1501 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
1561 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
1621 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
1681 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttctcgcggt
1741 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
1801 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
1861 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
1921 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
1981 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
2041 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
2101 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
2161 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
2221 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
2281 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
2341 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
2401 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
2461 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
2521 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
2581 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
2641 agcaacgcgg cctttttacg gttcctggca gatcctagat gtggcgcaac gatgccggcg
2701 acaagcagga gcgcaccgac ttcttccgca tcaagtgttt tggctctcag gccgaggccc
2761 acggcaagta tttgggcaag gggtcgctgg tattcgtgca gggcaagatt cggaatacca
2821 agtacgagaa ggacggccag acggtctacg ggaccgactt cattgccgat aaggtggatt
2881 atctggacac caaggcacca ggcgggtcaa atcaggaata agggcacatt gccccggcgt
2941 gagtcggggc aatcccgcaa ggagggtgaa tgaatcggac gtttgaccgg aaggcataca
3001 ggcaagaact gatcgacgcg gggttttccg ccgaggatgc cgaaaccatc gcaagccgca
3061 ccgtcatgcg tgcgccccgc gaaaccttcc agtccgtcgg ctcgatggtc cagcaagcta
3121 cggccaagat cgagcgcgac agcgtgcaac tggctccccc tgccctgccc gcgccatcgg
3181 ccgccgtgga gcgttcgcgt cgtcttgaac aggaggcggc aggtttggcg aagtcgatga
3241 ccatcgacac gcgaggaact atgacgacca agaagcgaaa aaccgccggc gaggacctgg
3301 caaaacaggt cagcgaggcc aagcaggccg cgttgctgaa acacacgaag cagcagatca
3361 aggaaatgca gctttccttg ttcgatattg cgccgtggcc ggacacgatg cgagcgatgc
3421 caaacgacac ggcccgctct gccctgttca ccacgcgcaa caagaaaatc ccgcgcgagg
3481 cgctgcaaaa caaggtcatt ttccacgtca acaaggacgt gaagatcacc tacaccggcg
3541 tcgagctgcg ggccgacgat gacgaactgg tgtggcagca ggtgttggag tacgcgaagc
3601 gcacccctat cggcgagccg atcaccttca cgttctacga gctttgccag gacctgggct
3661 ggtcgatcaa tggccggtat tacacgaagg ccgaggaatg cctgtcgcgc ctacaggcga
3721 cggcgatggg cttcacgtcc gaccgcgttg ggcacctgga atcggtgtcg ctgctgcacc
3781 gcttccgcgt cctggaccgt ggcaagaaaa cgtcccgttg ccaggtcctg atcgacgagg
3841 aaatcgtcgt gctgtttgct ggcgaccact acacgaaatt catatgggag aagtaccgca
3901 agctgtcgcc gacggcccga cggatgttcg actatttcag ctcgcaccgg gagccgtacc
3961 cgctcaagct ggaaaccttc cgcctcatgt gcggatcgga ttccacccgc gtgaagaagt
4021 ggcgcgagca ggtcggcgaa gcctgcgaag agttgcgagg cagcggcctg gtggaacacg
4081 cctgggtcaa tgatgacctg gtgcattgca aacgctaggg ccttgtgggg tcagttccgg
4141 ctgggggttc agcagccagc gcctgatctg gggaaccctg tggttggcat gcacatacaa
4201 atggacgaac ggataaacct tttcacgccc ttttaaatat ccgattattc taataaacgc
4261 tcttttctct taggtttacc cgccaatata tcctgtcaaa cactgatagt ttaaaccact
4321 tcgtgcagaa gacaattgca gcgtgagacc gtcacagctt gtctgtaagc ggatgccggg
4381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
4441 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
4501 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
4561 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
4621 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
4681 acggccagtg aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
4741 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
4801 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
4861 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcggcaa acctgtcgtg
4921 ccagctgcgg tctcactccg gatccgaatt cggcattgtc ttcacaga
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