LOCUS       sequence-441207-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for FusX-based assembly of alphaDdCBE arm with 
            DddAtox 1397N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  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 6561)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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: FusX alphaDdCBE 1397N
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     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-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..981
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             994..1059
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(1486..1779)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCS"
     primer_bind     1629..1651
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1643..1660
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1644..1660
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1670..1688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1736..1756)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1736..1754)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1775..1791)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1799..1815
                     /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(1823..1829,1830..1847,1848..1853))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1868..1889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2345..2596
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      2793..3221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2880..2899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3090..3111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3230..3250)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3235..3564
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3415..3550
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3477..3496
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3612..3659
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3678..4076
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4234..4355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4271..4290)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4325..4344
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4404..4420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4428..4444
                     /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(4452..4458,4459..4476,4477..4482))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4497..4518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4635..4652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4806..5394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4886..4905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5565..6356,6357..6425))
                     /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     6188..6207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6426..6530)
                     /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 ctggagacgg gcgccttagc tacagggcgc
     1501 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     1561 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     1621 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     1681 actatagggc gaattgggta ccgggccccc cctcgaggtc ctccagcttt tgttcccttt
     1741 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1801 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1861 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccacc
     1921 ggtcgtctcc aacgatcacc tggtggccct ggcctgcctg ggcggtcgtc cggccctgga
     1981 tgcggtgaag aaaggcctgg gtggatccgg cagctacgcc ctgggtccgt atcagattag
     2041 cgccccgcag ctgccagctt acaatggtca gaccgtgggt accttctact atgtgaacga
     2101 cgcgggcggt ctggagagca aggtgtttag cagcggcggt ccaaccccgt acccaaacta
     2161 tgccaatgcc ggtcatgtgg agggtcagag cgccctgttc atgcgtgata acggcatcag
     2221 cgagggtctg gtgttccaca acaacccgga aggcacctgc ggtttttgcg tgaacatgac
     2281 cgagaccctg ctgccggaaa acgcgaaaat gaccgtggtg ccgccggaag gttctggcgg
     2341 ctcaactaat ctgagcgaca tcattgagaa ggagactggg aaacagctgg tcattcagga
     2401 gtccatcctg atgctgcctg aggaggtgga ggaagtgatc ggcaacaagc cagagtctga
     2461 catcctggtg cacaccgcct acgacgagtc cacagatgag aatgtgatgc tgctgacctc
     2521 tgacgccccc gagtataagc cttgggccct ggtcatccag gattctaacg gcgagaataa
     2581 gatcaagatg ctgtgataag gggtctttgt cctctgtact gtctctctcc ttgcccctaa
     2641 cccaaaaagc ttcatttttc tgtgtaggct gcacaagagc cttgattgaa gatatattct
     2701 ttctgaacag tatttaaggt ttccaataaa atgtacaccc ctcagaactt ctgaggcgga
     2761 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2821 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2881 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2941 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3001 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3061 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3121 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3181 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3241 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3301 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3361 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3421 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3481 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3541 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3601 gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     3661 aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     3721 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     3781 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     3841 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     3901 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     3961 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     4021 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagcac
     4081 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc
     4141 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4201 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4261 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4321 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4381 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4441 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4501 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4561 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4621 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4861 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4921 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4981 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5041 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5101 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5161 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5221 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5281 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5341 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5401 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5461 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5521 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgtttaatc
     5581 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5641 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5701 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5761 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5821 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5881 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5941 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6001 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6061 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6121 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6181 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6241 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6301 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6361 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6421 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6481 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6541 cgaaaagtgc cacctgacgt c
//
