LOCUS       Exported                7646 bp ds-DNA     circular SYN 22-JAN-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7646)
  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..7646
                     /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..2964
                     /codon_start=1
                     /product="5'T caatagagggggaaa 3'"
                     /label=ATP6 TALE T2
                     /translation="LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIAS
                     NIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLT
                     PDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLP
                     VLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQA
                     LETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAI
                     ASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG
                     LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL
                     LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK
                     QALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALE"
     CDS             2965..3087
                     /codon_start=1
                     /label=TALE CTD
                     /translation="SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLG"
     primer_bind     complement(3072..3091)
                     /label=FusX R
     CDS             3088..3093
                     /codon_start=1
                     /label=Linker
                     /translation="GS"
     CDS             3094..3417
                     /codon_start=1
                     /label=DddAtox 1397N
                     /translation="GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGP
                     TPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVV
                     PPEG"
     CDS             3418..3429
                     /codon_start=1
                     /label=Linker
                     /translation="SGGS"
     CDS             3430..3681
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     misc_feature    3685..3832
                     /label=ATP5B 3'UTR
     rep_origin      3878..4306
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4320..4649
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4500..4635
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4697..4744
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4763..5161
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5319..5440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5356..5375)
                     /label=SV40pA-R
                     /note="20"
     primer_bind     complement(5489..5505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5489..5505)
                     /label=M13R
                     /note="17"
     primer_bind     complement(5503..5522)
                     /label=M13-48REV
                     /note="20"
     protein_bind    5513..5529
                     /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(5537..5567)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5582..5603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5891..6479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6650..7510)
                     /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(7511..7615)
                     /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 atcgccagcc acgatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1561 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacattggc
     1621 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1681 ctgaccccgg accaagtggt ggctatcgcc agcaacattg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1921 attggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1981 catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg
     2041 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     2101 caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2161 ctgttgccgg tgctgtgcca ggaccatggc ctcacacccg atcaggtcgt ggctatcgcc
     2221 agcaacaatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacaa tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagcaac aatggcggca 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 agcaacattg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2821 gtggctatcg ccagcaacat tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     2941 attggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc tgatccggcg
     3001 ttggccgcgt tgaccaacga tcacctggtg gccctggcct gcctgggcgg tcgtccggcc
     3061 ctggatgcgg tgaagaaagg cctgggtgga tccggcagct acgccctggg tccgtatcag
     3121 attagcgccc cgcagctgcc agcttacaat ggtcagaccg tgggtacctt ctactatgtg
     3181 aacgacgcgg gcggtctgga gagcaaggtg tttagcagcg gcggtccaac cccgtaccca
     3241 aactatgcca atgccggtca tgtggagggt cagagcgccc tgttcatgcg tgataacggc
     3301 atcagcgagg gtctggtgtt ccacaacaac ccggaaggca cctgcggttt ttgcgtgaac
     3361 atgaccgaga ccctgctgcc ggaaaacgcg aaaatgaccg tggtgccgcc ggaaggttct
     3421 ggcggctcaa ctaatctgag cgacatcatt gagaaggaga ctgggaaaca gctggtcatt
     3481 caggagtcca tcctgatgct gcctgaggag gtggaggaag tgatcggcaa caagccagag
     3541 tctgacatcc tggtgcacac cgcctacgac gagtccacag atgagaatgt gatgctgctg
     3601 acctctgacg cccccgagta taagccttgg gccctggtca tccaggattc taacggcgag
     3661 aataagatca agatgctgtg ataaggggtc tttgtcctct gtactgtctc tctccttgcc
     3721 cctaacccaa aaagcttcat ttttctgtgt aggctgcaca agagccttga ttgaagatat
     3781 attctttctg aacagtattt aaggtttcca ataaaatgta cacccctcag aacttctgag
     3841 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3901 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3961 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4021 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4081 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4141 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4201 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4261 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     4321 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4381 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4441 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4501 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4561 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4621 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4681 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     4741 cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat ccaccctcat
     4801 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     4861 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     4921 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     4981 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     5041 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     5101 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     5161 agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg
     5221 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5281 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5341 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5401 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5461 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5521 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5581 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5641 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5701 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5761 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5821 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5881 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5941 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6001 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6061 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6121 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6181 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6241 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6301 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6361 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6421 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6481 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6541 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6601 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgtt
     6661 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6721 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6781 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6841 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6901 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6961 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7021 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7081 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7141 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7201 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7261 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7321 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7381 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7441 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7501 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7621 ttccccgaaa agtgccacct gacgtc
//
