LOCUS       Exported                6327 bp ds-DNA     circular SYN 14-JAN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  AUTHORS   Mayo Clinic Research
  TITLE     Direct Submission
  JOURNAL   Exported Friday, May 24, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6327
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        267..646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        647..850
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     801..821
                     /label=CMV-Forward
                     /note="21"
     primer_bind     895..914
                     /label=T7
                     /note="20"
     promoter        895..913
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             936..938
                     /codon_start=1
                     /label=START
                     /translation="M"
     CDS             942..1025
                     /codon_start=1
                     /label=COX8A MTS
                     /translation="SVLTPLLLRGLTGSARRLPVPRAKIHSL"
     CDS             1026..1091
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1095..1505
                     /codon_start=1
                     /product="Adapted from Bacman S. et al., 2013 to recognize 
                     C over T at the N0 position.This is NT-alpha-N from the 
                     paper ""Directed evolution of the TALE N-terminal domain 
                     for recognition of all 5' bases"" by Lamb et al. This TALE 
                     NTD variant accepts substrates with any 5' residue and 
                     maintains high activity.They took this seemingly convoluted
                     approach because they used the mitoTALE that Bacman S. et 
                     al. used in their 2013 study, which was designed to 
                     recognize the m.14459G>A mutation. The mutation itself 
                     helps fulfill the 5'-T constraint. Since in the DdCBE paper
                     the authors were inducing de novo mutagenesis, they needed 
                     to target the wild-type sequence. They knew that those RVDs
                     worked, so they simply changed the TALE NTD so that it 
                     could bind the wild-type sequence. So it wasn't convoluted,
                     it was actually smart."
                     /label=TALE NTD NT-alpha-N'
                     /translation="DIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA
                     LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQ
                     LDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL"
     primer_bind     1303..1322
                     /label=FusX F
     misc_feature    1335..1343
                     /label=NT-alpha-N motif
     CDS             complement(1518..1811)
                     /codon_start=1
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCS"
     primer_bind     1676..1692
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1702..1721
                     /label=T7
                     /note="20"
     promoter        1702..1720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1768..1786)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1807..1823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1807..1823)
                     /label=M13R
                     /note="17"
     primer_bind     complement(1821..1840)
                     /label=M13-48REV
                     /note="20"
     protein_bind    1831..1847
                     /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(1855..1885)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1900..1921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1963..2034
                     /codon_start=1
                     /label=TALE CTD'
                     /translation="NDHLVALACLGGRPALDAVKKGLG"
     primer_bind     complement(2019..2038)
                     /label=FusX R
     CDS             2035..2040
                     /codon_start=1
                     /label=Linker
                     /translation="GS"
     CDS             2041..2130
                     /codon_start=1
                     /label=DddAtox 1397C
                     /translation="AIPVKRGATGETKVFTGNSNSPKSPTKGGC"
     CDS             2131..2142
                     /codon_start=1
                     /label=Linker
                     /translation="SGGS"
     CDS             2143..2394
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     misc_feature    2398..2545
                     /label=ATP5B 3'UTR
     rep_origin      2591..3019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3033..3362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3213..3348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3410..3457
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3476..3874
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4032..4153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4069..4088)
                     /label=SV40pA-R
                     /note="20"
     primer_bind     complement(4202..4218)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4202..4218)
                     /label=M13R
                     /note="17"
     primer_bind     complement(4216..4235)
                     /label=M13-48REV
                     /note="20"
     protein_bind    4226..4242
                     /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(4250..4280)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4295..4316
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4604..5192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5363..6223)
                     /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"
     promoter        complement(join(6224..6327,1))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
       61 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
      121 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
      181 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      241 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      301 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      361 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      421 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      481 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      541 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      601 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      661 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      721 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      781 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      841 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      901 gactcactat agggagaccc aagctggcta gcaccatggc ctccgttctg accccgctgc
      961 tgctgcgtgg cctgaccggc tccgctcgtc gtctgccagt tccgcgtgcg aaaatccatt
     1021 ccctggacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     1081 acgatgacaa gatggacatc gcggatctgc gtaccctggg ttacagccag cagcagcagg
     1141 agaagatcaa gccaaaggtg cgcagcaccg tggcccagca ccatgaagct ctggtgggtc
     1201 acggcttcac ccacgcgcac atcgtggctc tgagccagca cccagccgcg ctgggtaccg
     1261 tggccgtgaa atatcaggac atgattgctg ccctgccaga ggctacccat gaagcgattg
     1321 tgggtgtggg caagagagga gccggtgctc gtgcgctgga ggccctgctg accgtggccg
     1381 gtgaactgcg tggcccgccg ctgcagctgg ataccggcca gctgctgaaa atcgcgaaac
     1441 gtggcggtgt gaccgctgtg gaagctgtgc atgcctggcg taatgctctg accggtgccc
     1501 ccctggagac gggcgcctta gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     1561 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     1621 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     1681 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
     1741 cccctcgagg tcctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     1801 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     1861 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     1921 aattgcgttg cgctcactgc ccgctttcca ccggtcgtct ccaacgatca cctggtggcc
     1981 ctggcctgcc tgggcggtcg tccggccctg gatgcggtga agaaaggcct gggtggatcc
     2041 gccattccag tgaagcgcgg cgctaccggt gaaaccaaag tgtttaccgg taacagcaac
     2101 agcccgaaga gcccgaccaa aggcggttgc tctggcggct caactaatct gagcgacatc
     2161 attgagaagg agactgggaa acagctggtc attcaggagt ccatcctgat gctgcctgag
     2221 gaggtggagg aagtgatcgg caacaagcca gagtctgaca tcctggtgca caccgcctac
     2281 gacgagtcca cagatgagaa tgtgatgctg ctgacctctg acgcccccga gtataagcct
     2341 tgggccctgg tcatccagga ttctaacggc gagaataaga tcaagatgct gtgataaggg
     2401 gtctttgtcc tctgtactgt ctctctcctt gcccctaacc caaaaagctt catttttctg
     2461 tgtaggctgc acaagagcct tgattgaaga tatattcttt ctgaacagta tttaaggttt
     2521 ccaataaaat gtacacccct cagaacttct gaggcggaaa gaaccagctg gggctctagg
     2581 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2641 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2701 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2761 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2821 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2881 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2941 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3001 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3061 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3121 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3181 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3241 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3301 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3361 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     3421 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     3481 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     3541 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     3601 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     3661 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     3721 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     3781 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     3841 attgctgccc tctggttatg tgtgggaggg ctaagcactt cgtggccgag gagcaggact
     3901 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3961 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4021 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4141 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4201 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4261 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5041 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5281 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5341 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5401 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5461 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5521 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5581 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5641 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5701 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5761 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5821 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5881 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5941 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6001 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6061 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6121 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6181 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6241 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6301 agaaaaataa acaaataggg gttccgc
//
