LOCUS       Exported                5913 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  for e. coli expression, Golden Gate Compatible TALEN incorporated 
            with N Terminal 6xHIS Tag, Codon Optimized FokI.
ACCESSION   .
VERSION     .
KEYWORDS    pET-HIS-Sangamo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5913)
  TITLE     Bao Lab Golden Gate plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5913)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5913
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     925..944
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(964..1041)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1247..1266
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1350..1368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1369..1393
                     /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)."
     RBS             1408..1430
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1449..1466
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1476..1493
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1500..1565
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1572..1592
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1749..2324)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TGERIAPGVHCLYRCYSPSLRDLQQLPGVELERRPPKLTRHSQQRLKCSRSRPLFTDPY
                     NCLVRGFGQGRNHILVFDSHRPKRRRVL"
     primer_bind     2174..2196
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2188..2205
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2189..2205
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2215..2233
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2281..2301)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2281..2299)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2320..2336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2320..2336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2333..2355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2344..2360
                     /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(2368..2398)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2413..2434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2620..3207
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             3251..3268
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3321..3339)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3335..3382
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3419..3874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3506..3525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3716..3737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3967..4782)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4690..4709
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4774..4793
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4904..5492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5393..5412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5646..5663
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
       61 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
      121 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
      181 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
      241 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
      301 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
      361 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
      421 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
      481 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
      541 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
      601 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
      661 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
      721 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
      781 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
      841 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
      901 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
      961 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     1021 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     1081 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     1141 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     1201 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     1261 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     1321 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     1441 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg
     1501 actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg
     1561 acaagatggc ccccaagaag aagaggaagg tgggcattca ccgcggggta cctatggtgg
     1621 acttgaggac actcggttat tcgcaacagc aacaggagaa aatcaagcct aaggtcagga
     1681 gcaccgtcgc gcaacaccac gaggcgcttg tggggcatgg cttcactcat gcgcatattg
     1741 tcgcgctttc acagcaccct gcggcgcttg ggacggtggc tgtcaaatac caagatatga
     1801 ttgcggccct gcccgaagcc acgcacgagg caattgtagg ggtcggtaaa cagtggtcgg
     1861 gagcgcgagc acttgaggcg ctgctgactg tggcgggtga gcttaggggg cctccgctcc
     1921 agctcgacac cgggcagctg ctgaagatcg cgaagagagg gggagtaaca gcggtagagg
     1981 cagtgcacgc ctggcgcaat gcgctcaccg gtgcccccct ggagacgggc gccgctacag
     2041 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2101 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2161 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     2221 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc
     2281 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2341 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2401 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2461 ccaccggtcg tctccaacga ccatctggtg gcgttggcat gtcttggtgg acgacccgcg
     2521 ctcgatgcag tcaaaaaggg tctgcctcat gctcccgcat tgatcaaaag aaccaaccgg
     2581 cggattcccg agagaacttc ccatcgagtc gcgggatccc agctggtgaa gagcgagctg
     2641 gaggagaaga agtccgagct gcggcacaag ctgaagtacg tgccccacga gtacatcgag
     2701 ctgatcgaga tcgccaggaa cagcacccag gaccgcatcc tggagatgaa ggtgatggag
     2761 ttcttcatga aggtgtacgg ctacagggga aagcacctgg gcggaagcag aaagcctgac
     2821 ggcgccatct atacagtggg cagccccatc gattacggcg tgatcgtgga cacaaaggcc
     2881 tacagcggcg gctacaatct gcctatcggc caggccgacg agatgcagag atacgtggag
     2941 gagaaccaga cccggaataa gcacatcaac cccaacgagt ggtggaaggt gtaccctagc
     3001 agcgtgaccg agttcaagtt cctgttcgtg agcggccact tcaagggcaa ctacaaggcc
     3061 cagctgacca ggctgaacca catcaccaac tgcaatggcg ccgtgctgag cgtggaggag
     3121 ctgctgatcg gcggcgagat gatcaaagcc ggcaccctga cactggagga ggtgcggcgc
     3181 aagttcaaca acggcgagat caacttctga gaattcgagc tccgtcgaca agcttgcggc
     3241 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     3301 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     3361 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     3421 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3481 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3541 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3601 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3661 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3721 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3781 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3841 gcgaatttta acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg
     3901 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3961 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4021 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     4081 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     4141 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     4201 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     4261 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     4321 tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     4381 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4441 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4501 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4561 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4621 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4681 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4741 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4801 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     4861 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4921 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4981 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5041 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5101 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5161 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5221 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5281 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5341 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5401 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5461 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5521 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5581 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5641 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     5701 cgcatctgtg cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5761 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5881 tttgcgtatt gggcgccagg gtggtttttc ttt
//
