LOCUS       Exported                7412 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 7412)
  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..7412
                     /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"
     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 aggcctacccgccgc 3'"
                     /label=ATP6 TALE T1
                     /translation="LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIAS
                     NNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLT
                     PDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLP
                     VLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQA
                     LETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAI
                     ASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG
                     LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRL
                     LPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGK
                     QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALE"
     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..3183
                     /codon_start=1
                     /label=DddAtox 1397C
                     /translation="AIPVKRGATGETKVFTGNSNSPKSPTKGGC"
     CDS             3184..3195
                     /codon_start=1
                     /label=Linker
                     /translation="SGGS"
     CDS             3196..3447
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     misc_feature    3451..3598
                     /label=ATP5B 3'UTR
     rep_origin      3644..4072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4086..4415
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4266..4401
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4463..4510
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4529..4927
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5085..5206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5122..5141)
                     /label=SV40pA-R
                     /note="20"
     primer_bind     complement(5255..5271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5255..5271)
                     /label=M13R
                     /note="17"
     primer_bind     complement(5269..5288)
                     /label=M13-48REV
                     /note="20"
     protein_bind    5279..5295
                     /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(5303..5333)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5348..5369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5657..6245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6416..7276)
                     /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(7277..7381)
                     /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 ctgaccccgg accaagtggt ggctatcgcc agcaacaatg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat cgccagccac
     1921 gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1981 catggcctga ccccggacca agtggtggct atcgccagca acggtggcgg caagcaagcg
     2041 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     2101 caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2161 ctgttgccgg tgctgtgcca ggaccatggc ctcacacccg atcaggtcgt ggctatcgcc
     2221 agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgactcc ggaccaagtg gtggctatcg ccagccacga tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagccac gatggcggca agcaagcgct cgaaacggtg
     2461 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2521 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2581 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     2641 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2701 ctgaccccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2821 gtggctatcg ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagccac
     2941 gatggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc tgatccggcg
     3001 ttggccgcgt tgaccaacga tcacctggtg gccctggcct gcctgggcgg tcgtccggcc
     3061 ctggatgcgg tgaagaaagg cctgggtgga tccgccattc cagtgaagcg cggcgctacc
     3121 ggtgaaacca aagtgtttac cggtaacagc aacagcccga agagcccgac caaaggcggt
     3181 tgctctggcg gctcaactaa tctgagcgac atcattgaga aggagactgg gaaacagctg
     3241 gtcattcagg agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag
     3301 ccagagtctg acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg
     3361 ctgctgacct ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac
     3421 ggcgagaata agatcaagat gctgtgataa ggggtctttg tcctctgtac tgtctctctc
     3481 cttgccccta acccaaaaag cttcattttt ctgtgtaggc tgcacaagag ccttgattga
     3541 agatatattc tttctgaaca gtatttaagg tttccaataa aatgtacacc cctcagaact
     3601 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3661 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3721 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3781 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3841 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3901 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3961 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4021 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     4081 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4141 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4201 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4261 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4321 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4381 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4441 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     4501 ataatacgac aaggtgagga actaaaccat ggccaagcct ttgtctcaag aagaatccac
     4561 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     4621 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     4681 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     4741 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     4801 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     4861 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     4921 gggctaagca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
     4981 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5041 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5101 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5161 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5221 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5281 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5341 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5401 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5461 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5521 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5581 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5641 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5701 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5761 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5821 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5881 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5941 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6001 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6061 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6121 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6181 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6241 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6301 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6361 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6421 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6481 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6541 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6601 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6661 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6721 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6781 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6841 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6901 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6961 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7021 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7081 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7141 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7201 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7261 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7321 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7381 gcacatttcc ccgaaaagtg ccacctgacg tc
//
