LOCUS Exported 6441 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Destination vector for Voytas Golden Gate TALEN assembly incorporating a CAG promoter for mammalian expression and a T7 promoter for synthesis of TALEN mRNA. Uses heterodimeric FokI domain (ELD).. ACCESSION . VERSION . KEYWORDS pCAG-T7-TALEN(Sangamo)-FokI-ELD-Destination SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6441) AUTHORS Hermann M, Cermak T, Voytas DF, Pelczar P TITLE Mouse genome engineering using designer nucleases. JOURNAL J Vis Exp. 2014 Apr 2;(86). doi: 10.3791/50930. PUBMED 24747757 REFERENCE 2 (bases 1 to 6441) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6441 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 12..42 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 50..66 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 74..90 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" enhancer 110..489 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 491..768 /label=chicken beta-actin promoter intron 769..1777 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1701..1723 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" promoter 1790..1808 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1828..1848 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(2152..2559) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG TRQCIARCMHCLHCGHAATFCNLHKLACVQL" primer_bind 2424..2440 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2450..2468 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2516..2536) /label=T3 /note="T3 promoter, forward primer" promoter complement(2516..2534) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2555..2571) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2579..2595 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(2603..2633) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2648..2669 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3464..3481) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3470..3694 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3766..3782) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 3924..4379 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4011..4030) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4221..4242 /label=F1ori-F /note="F1 origin, forward primer" promoter 4405..4509 /gene="bla" /label=AmpR promoter CDS 4510..5370 /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 complement(4728..4747) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5541..6129 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6030..6049 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6283..6300 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6417..6438 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 61 taacaatttc acacaggaaa cagctatgac catgaggcgc gccggattcg acattgatta 121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 481 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc 541 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg 601 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 661 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 721 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg 781 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg 841 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 901 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc 961 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt 1021 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg 1081 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt 1141 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc 1201 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg 1261 ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg gggctcgccg 1321 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg 1381 gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc gaggcgcggc 1441 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc 1501 ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg 1561 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1621 gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg gctgccttcg 1681 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc 1741 tgctaaccat gttcatgcct tcttcttttt cctacagatc cttaattaat aatacgactc 1801 actatagggg ccgccaccat gagatctcct aagaaaaaga ggaaggtgca ggtggatcta 1861 cgcacgctcg gctacagtca gcagcagcaa gagaagatca aaccgaaggt gcgttcgaca 1921 gtggcgcagc accacgaggc actggtgggc catgggttta cacacgcgca catcgttgcg 1981 ctcagccaac acccggcagc gttagggacc gtcgctgtca cgtatcagca cataatcacg 2041 gcgttgccag aggcgacaca cgaagacatc gttggcgtcg gcaaacagtg gtccggcgca 2101 cgcgccctgg aggccttgct cacggatgcg ggggagttga gaggtccgcc gttacagttg 2161 gacacaggcc aacttgtgaa gattgcaaaa cgtggcggcg tgaccgcaat ggaggcagtg 2221 catgcatcgc gcaatgcact gacgggtgcc cccctggaga cgggcgccgc tacagggcgc 2281 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 2341 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 2401 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc 2461 actatagggc gaattgggta ccgggccccc cctcgaggtc ctccagcttt tgttcccttt 2521 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 2581 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 2641 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccacc 2701 ggtcgtctcc aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga 2761 tgcagtgaaa aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat 2821 tggcgaacgc acgtcccatc gcgttgcggg atcccagctg gtgaagagcg agctggagga 2881 gaagaagtcc gagctgcggc acaagctgaa gtacgtgccc cacgagtaca tcgagctgat 2941 cgagatcgcc aggaacccca cccaggaccg catcctggag atgaaggtga tggagttctt 3001 catgaaggtg tacggctaca ggggagagca cctgggcgga agcagaaagc ctgacggcgc 3061 catctataca gtgggcagcc ccatcgatta cggcgtgatc gtggacacaa aggcctacag 3121 cggcggctac aatctgccta tcggccaggc cgacgagatg gagagatacg tggaggagaa 3181 ccagacacgg gataagcacc tcaaccccaa cgagtggtgg aaggtgtacc ctagcagcgt 3241 gaccgagttc aagttcctgt tcgtgagcgg ccacttcaag ggcaactaca aggcccagct 3301 gaccaggctg aaccacatca ccaactgcaa tggcgccgtg ctgagcgtgg aggagctgct 3361 gatcggcggc gagatgatca aagccggcac cctgacactg gaggaggtgc ggcgcaagtt 3421 caacaacggc gagatcaact tctgatagga gctcgctgat cagcctcgac tgtgccttct 3481 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 3541 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 3601 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 3661 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg 3721 ggctcgagat ccactagttc tagcctcgag gctagagcgg ccgccactgg ccgtcgtttt 3781 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 3841 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 3901 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 3961 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 4021 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 4081 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 4141 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 4201 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 4261 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 4321 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta 4381 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 4441 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 4501 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 4561 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 4621 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 4681 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 4741 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 4801 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 4861 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 4921 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 4981 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 5041 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 5101 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 5161 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 5221 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 5281 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 5341 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 5401 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 5461 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 5521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 5581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 5641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 5701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 5761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 5821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 5881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 5941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6181 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 6241 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 6301 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 6361 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 6421 gtgagttagc tcactcatta g //