LOCUS       Exported                7748 bp ds-DNA     circular SYN 02-NOV-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  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..7748
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-Forward
                     /note="21"
     primer_bind     863..882
                     /label=T7
                     /note="20"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    898..903
                     /label=Kozak sequence
     CDS             904..906
                     /codon_start=1
                     /label=START
                     /translation="M"
     CDS             910..993
                     /codon_start=1
                     /label=COX8A MTS
                     /translation="SVLTPLLLRGLTGSARRLPVPRAKIHSL"
     CDS             994..1059
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1063..1476
                     /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
                     LDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN"
     primer_bind     1271..1290
                     /label=FusX F
     CDS             1477..3066
                     /codon_start=1
                     /label=ND2 right TALE
                     /translation="LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIAS
                     NNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT
                     PDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLP
                     VLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQA
                     LETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAI
                     ASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG
                     LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL
                     LPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGK
                     QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVV
                     AIASNIGGKQALE"
     CDS             3067..3189
                     /codon_start=1
                     /label=TALE CTD
                     /translation="SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG"
     primer_bind     complement(3174..3193)
                     /label=FusX R
     CDS             3190..3195
                     /codon_start=1
                     /label=Linker
                     /translation="GS"
     CDS             3196..3519
                     /codon_start=1
                     /label=DddAtox 1397-N
                     /translation="GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGP
                     TPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVV
                     PPEG"
     CDS             3520..3531
                     /codon_start=1
                     /label=Linker
                     /translation="SGGS"
     CDS             3532..3783
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     misc_feature    3787..3934
                     /label=ATP5B 3'UTR
     rep_origin      3980..4408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4422..4751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4602..4737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4799..4846
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4865..5263
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5421..5542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5458..5477)
                     /label=SV40pA-R
                     /note="20"
     primer_bind     complement(5591..5607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5591..5607)
                     /label=M13R
                     /note="17"
     primer_bind     complement(5605..5624)
                     /label=M13-48REV
                     /note="20"
     protein_bind    5615..5631
                     /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(5639..5669)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5684..5705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5993..6581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6752..7612)
                     /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(7613..7717)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggcct ccgttctgac cccgctgctg ctgcgtggcc tgaccggctc cgctcgtcgt
      961 ctgccagttc cgcgtgcgaa aatccattcc ctggactaca aagaccatga cggtgattat
     1021 aaagatcatg acatcgatta caaggatgac gatgacaaga tggacatcgc ggatctgcgt
     1081 accctgggtt acagccagca gcagcaggag aagatcaagc caaaggtgcg cagcaccgtg
     1141 gcccagcacc atgaagctct ggtgggtcac ggcttcaccc acgcgcacat cgtggctctg
     1201 agccagcacc cagccgcgct gggtaccgtg gccgtgaaat atcaggacat gattgctgcc
     1261 ctgccagagg ctacccatga agcgattgtg ggtgtgggca agagaggagc cggtgctcgt
     1321 gcgctggagg ccctgctgac cgtggccggt gaactgcgtg gcccgccgct gcagctggat
     1381 accggccagc tgctgaaaat cgcgaaacgt ggcggtgtga ccgctgtgga agctgtgcat
     1441 gcctggcgta atgctctgac cggtgccccc ctgaacctga ccccggacca agtggtggct
     1501 atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1561 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc
     1621 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1681 ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1921 aatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1981 catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg
     2041 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     2101 caagtggtgg ctatcgccag caacaatggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2161 ctgttgccgg tgctgtgcca ggaccatggc ctcacacccg atcaggtcgt ggctatcgcc
     2221 agcaacggtg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacgg tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct cgaaacggtg
     2461 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2521 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2581 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc
     2641 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2701 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2821 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     2941 ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     3001 catggcctga ccccggacca agtggtggct atcgccagca acattggcgg caagcaagcg
     3061 ctcgaaagca ttgtggccca gctgagccgg cctgatccgg cgttggccgc gttgaccaac
     3121 gatcacctgg tggccctggc ctgcctgggc ggtcgtccgg ccctggatgc ggtgaagaaa
     3181 ggcctgggtg gatccggcag ctacgccctg ggtccgtatc agattagcgc cccgcagctg
     3241 ccagcttaca atggtcagac cgtgggtacc ttctactatg tgaacgacgc gggcggtctg
     3301 gagagcaagg tgtttagcag cggcggtcca accccgtacc caaactatgc caatgccggt
     3361 catgtggagg gtcagagcgc cctgttcatg cgtgataacg gcatcagcga gggtctggtg
     3421 ttccacaaca acccggaagg cacctgcggt ttttgcgtga acatgaccga gaccctgctg
     3481 ccggaaaacg cgaaaatgac cgtggtgccg ccggaaggtt ctggcggctc aactaatctg
     3541 agcgacatca ttgagaagga gactgggaaa cagctggtca ttcaggagtc catcctgatg
     3601 ctgcctgagg aggtggagga agtgatcggc aacaagccag agtctgacat cctggtgcac
     3661 accgcctacg acgagtccac agatgagaat gtgatgctgc tgacctctga cgcccccgag
     3721 tataagcctt gggccctggt catccaggat tctaacggcg agaataagat caagatgctg
     3781 tgataagggg tctttgtcct ctgtactgtc tctctccttg cccctaaccc aaaaagcttc
     3841 atttttctgt gtaggctgca caagagcctt gattgaagat atattctttc tgaacagtat
     3901 ttaaggtttc caataaaatg tacacccctc agaacttctg aggcggaaag aaccagctgg
     3961 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4021 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4081 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4141 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4201 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4261 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4321 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4381 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     4441 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4501 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4561 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4621 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4681 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4741 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt
     4801 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     4861 aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     4921 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     4981 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     5041 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     5101 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga
     5161 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg
     5221 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc gtggccgagg
     5281 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5341 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5401 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5461 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5521 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5581 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5641 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5701 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5761 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5821 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5881 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5941 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6001 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6061 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6121 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6181 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6241 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6301 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6361 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6421 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6481 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6541 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6601 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6661 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6721 aaagtatata tgagtaaact tggtctgaca gttaccaatg tttaatcagt gaggcaccta
     6781 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6841 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6901 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6961 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7021 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7081 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7141 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7201 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7261 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7321 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7381 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7441 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7501 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7561 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7621 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7681 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7741 ctgacgtc
//
