LOCUS Exported 7609 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pTAL plasmid for cloning of megaTALs or other TAL-fusions and expression in mammalian cells. RVDs are cloned between Age-Xho sites and meganuclease/fusion is cloned between Xba-Sal sites. . ACCESSION . VERSION . KEYWORDS pCVL TAL(Nd154C63) SFFV Age-Xho/Xba-Sal linkers SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7609) AUTHORS Boissel S, Jarjour J, Astrakhan A, Adey A, Gouble A, Duchateau P, Shendure J, Stoddard BL, Certo MT, Baker D, Scharenberg AM TITLE megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. JOURNAL Nucleic Acids Res. 2013 Nov 26. PUBMED 24285304 REFERENCE 2 (bases 1 to 7609) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7609 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 9..235 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 236..416 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 463..588 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1080..1313 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1498..1542 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 1809..1926 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2621..3022 /label=SFFV promoter /note="spleen focus-forming virus long terminal repeat (LTR) promoter" CDS 3027..3728 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MASELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY CDLPSKLGHKLN" CDS 3732..3785 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 3792..3818 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3822..3842 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind 5148..5181 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(5386..5403) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5392..5616 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5689..5706) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5860..6448) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5940..5959) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6619..7479) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7242..7261 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7480..7584) /gene="bla" /label=AmpR promoter ORIGIN 1 gacgtcaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta 61 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg 121 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac 181 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc 241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg 421 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact 481 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa 541 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 601 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat 661 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 721 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 781 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 841 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 901 aaaagtaaga ccaccgcaca gcaagcggcc ctgatcttca gacctggagg aggagatatg 961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1381 aatgaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 1921 aattttatcg attacgcgtg tggcatctga agcaccacca gcgagcgaga gctagagaga 1981 aggaaagcca ccgacttcac cgcctccgag ctgctccggg tcgcgggtct gcagcgtcta 2041 cggccctccg cgcctacagc tcaagccaca tccgaagggg gagggagccg ggagctgcgc 2101 gcggggccgc cggggggagg ggtggcaccg cccacgccgg gcggccacga agggcggggc 2161 agcgggcgcg cgcccggcgg ggggaggggc cgcgcgccgc gcccgctggg aattggggcc 2221 ctagggggag ggcggaggcg ccgacgaccg cggcacttac cgttcgcggc gtggcgcccg 2281 gtggtcccca aggggaggga agggggaggc ggggcgagga cagtgaccgg agtctcctca 2341 gcggtggctt ttctgcttgg cagcctcagc ggctggcgcc aaaaccggac tccgcccact 2401 tcctcgcccc tgcggtgcga gggtgtggaa tcctccagac gctgggggag ggggagttgg 2461 gagcttaaaa actagtaccc ctttgggacc actttcagca gcgaactctc ctgtacacca 2521 ggggtcagtt ccacagacgc gggccagggg tgggtcattg cggcgtgaac aataatttga 2581 ctagaagttg attcgggtgt ttccggaatt cctagctgca gtaacgccat tttgcaaggc 2641 atggaaaaat accaaaccaa gaatagagaa gttcagatca agggcgggta catgaaaata 2701 gctaacgttg ggccaaacag gatatctgcg gtgagcagtt tcggccccgg cccggggcca 2761 agaacagatg gtcaccgcag tttcggcccc ggcccgaggc caagaacaga tggtccccag 2821 atatggccca accctcagca gtttcttaag acccatcaga tgtttccagg ctcccccaag 2881 gacctgaaat gaccctgcgc cttatttgaa ttaaccaatc agcctgcttc tcgcttctgt 2941 tcgcgcgctt ctgcttcccg agctctataa aagagctcac aacccctcac tcggcgcgcc 3001 agtcctccga cagactgagt cgcccgatgg ctagcgagct gattaaggag aacatgcaca 3061 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg 3121 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc 3181 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca 3241 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg 3301 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc 3361 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc 3421 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacactg taccccgctg 3481 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga 3541 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg 3601 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg 3661 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca 3721 agcttaattc agagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatccgg 3781 gcccctgcag gtatccatat gatgtcccag attatgcgcc acctaagaag aaacgcaaag 3841 tcgtggatct acgcacgctc ggctacagtc agcagcagca agagaagatc aaaccgaagg 3901 tgcgttcgac agtggcgcag caccacgagg cactggtggg ccatgggttt acacacgcgc 3961 acatcgttgc gctcagccaa cacccggcag cgttagggac cgtcgctgtc acgtatcagc 4021 acataatcac ggcgttgcca gaggcgacac acgaagacat cgttggcgtc ggcaaacagt 4081 ggtccggcgc acgcgccctg gaggccttgc tcacggatgc gggggagttg agaggtccgc 4141 cgttacagtt ggacacaggc caacttgtga agattgcaaa acgtggcggc gtgaccgcaa 4201 tggaggcagt gcatgcatcg cgcaatgcac tgaccggtgc atgctagctg actgacctcg 4261 agagcattgt ggcccagctg agccggcctg atccggcgtt ggccgcgttg accaacgacc 4321 acctcgtcgc cttggcctgc ctcggcggac gtcctgccat ggatgcagtg aaaaagggat 4381 tgccgcacgc gccggaattg atcagaagag tcaatcgccg tattggcgaa cgcacgtccc 4441 atcgcgttgc gatatctaga tgagacgtag tgatctgacg tctcgtcgac cgagcatctt 4501 accgccattt atacccatat ttgttctgtt tttcttgatt tgggtataca tttaaatgtt 4561 aatagaacaa aatggtgggg caatcattta catttttagg gatatgtaat tactagttca 4621 ggtgtattgc cacaagacaa acatgttaag aaactttccc gttatttacg ctctgttcct 4681 gttaatcaac ctctggatta caaaatttgt gaaagattga ctgatattct taactatgtt 4741 gctcctttta cgctgtgtgg atatgctgct ttatagcctc tgtatctagc tattgcttcc 4801 cgtacggctt tcgttttctc ctccttgtat aaatcctggt tgctgtctct tttagaggag 4861 ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg tgtttgctga cgcaaccccc 4921 actggctggg gcattgccac cacctgtcaa ctcctttctg ggactttcgc tttccccctc 4981 ccgatcgcca cggcagaact catcgccgcc tgccttgccc gctgctggac aggggctagg 5041 ttgctgggca ctgataattc cgtggtgttg tcatcgaatt cggtaccttt ttaaaagaaa 5101 aggggggact ggaagggcta attcactccc aacgaagaca agatatcata acttcgtata 5161 gcatacatta tacgaagtta taatttattt gtgaaatttg tgatgctatt gctttatttg 5221 taaccatatg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattgcttt 5281 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 5341 tagggaaccc actgcttaag cctcaataaa gcttgcctcg accagcctcg actgtgcctt 5401 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5461 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 5521 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 5581 atagcaggca tgctggggat gcggtgggct ctatggcctg cagctgcatt aatgaatcgg 5641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 5701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 6001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 6061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 6121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 6181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 6241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6301 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 6421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 6541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 6601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 6661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 6721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 7021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 7081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 7141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 7201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 7261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 7441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 7501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 7561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacct //