LOCUS       sequence-438629-        5621 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses mitochondrially targeted TALE-free DddA6 1397N-UGI in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5621)
  AUTHORS   Castillo SR, Simone BW, Clark KJ, Devaux P, Ekker SC
  TITLE     Unconstrained Precision Mitochondrial Genome Editing with 
            alphaDdCBEs.
  JOURNAL   Hum Gene Ther. 2024 Sep 24. doi: 10.1089/hum.2024.073.
  PUBMED    39212664
REFERENCE   2  (bases 1 to 5621)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Hum Gene 
            Ther. 2024 Sep 24. doi: 10.1089/hum.2024.073."
COMMENT     Sequence Label: TALE-free DddA6 1397N
FEATURES             Location/Qualifiers
     source          1..5621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(76..95)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     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-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     895..914
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        895..913
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             936..1013
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1026..1091
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1437..1688
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      1885..2313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1972..1991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2182..2203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2322..2342)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2327..2656
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2507..2642
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2569..2588
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2704..2751
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2770..3168
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3326..3447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3363..3382)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3417..3436
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3496..3512)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3496..3512)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3509..3531)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3520..3536
                     /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(join(3544..3550,3551..3568,3569..3574))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3589..3610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3727..3744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3898..4486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3978..3997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4657..5448,5449..5517))
                     /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"
     primer_bind     5280..5299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5518..5621,1))
                     /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 gatgggatcc ggcagctacg ccctgggtcc gtatcagatt agcgccccgc
     1141 agctgccagc ttacaatggt cggaccgtgg gtaccttcta ctatgtgaac gacgcgggcg
     1201 gtctggagag caaggtgttt atcagcggcg gtccaacccc gtacccaaac tatgccaatg
     1261 ccggtcatgt ggagggtcag agcgccctgt tcatgcgtga taacggcatc agcgagggtc
     1321 tggtgttcca caacaacccg gaaggcacct gcggtttttg cgtgaacatg atcgagaccc
     1381 tgctgccgga aaacgcgaaa atgaccgtgg tgccgccgga aggttctggc ggctcaacta
     1441 atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag gagtccatcc
     1501 tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct gacatcctgg
     1561 tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc tctgacgccc
     1621 ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat aagatcaaga
     1681 tgctgtgata aggggtcttt gtcctctgta ctgtctctct ccttgcccct aacccaaaaa
     1741 gcttcatttt tctgtgtagg ctgcacaaga gccttgattg aagatatatt ctttctgaac
     1801 agtatttaag gtttccaata aaatgtacac ccctcagaac ttctgaggcg gaaagaacca
     1861 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1921 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1981 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2041 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2101 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2161 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2221 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2281 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     2341 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2401 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2461 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2521 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2581 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2641 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     2701 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     2761 aactaaacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     2821 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     2881 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     2941 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     3001 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     3061 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     3121 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaagc acttcgtggc
     3181 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     3241 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3301 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3361 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3421 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     3481 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3541 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3601 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3661 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3721 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3781 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3841 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3901 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3961 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4021 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4081 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4141 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4201 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4261 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4321 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4381 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4441 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4501 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4561 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4621 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4681 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4741 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4801 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4861 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4921 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4981 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5041 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5101 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5161 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5221 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5281 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5341 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5401 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5461 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5521 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5581 atttgaatgt atttagaaaa ataaacaaat aggggttccg c
//
