LOCUS       sequence-415656-        8624 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of codon-optimized dead mitochondrial base editor in 
            human cells; cell sorting; blasticidin drug selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8624)
  TITLE     Quantifying constraint in the human mitochondrial genome
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8624)
  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: 
            "Unpublished"
COMMENT     Sequence Label: m.3047-Left TALE-G1397-N-dead-mCherry
FEATURES             Location/Qualifiers
     source          1..8624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(109..128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        300..679
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        680..883
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     834..854
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     928..947
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        928..946
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             969..1046
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1059..1124
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3699..3947
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3957..4013
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     rep_origin      4921..5349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5008..5027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5218..5239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5358..5378)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5363..5692
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5543..5678
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5605..5624
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5740..5787
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5806..6204
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6362..6483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6399..6418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6453..6472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6532..6548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6532..6548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6545..6567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6556..6572
                     /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(6580..6586,6587..6604,6605..6610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6625..6646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6763..6780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6934..7522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7014..7033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7693..8484,8485..8553))
                     /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     8316..8335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8554..8624,1..34))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
       61 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      121 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      181 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      241 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      301 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      361 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      421 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      481 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      541 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      601 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      661 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      721 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      781 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      841 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      901 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      961 ctagcaccat ggcctccgtt ctgaccccgc tgctgctgcg tggcctgacc ggctccgctc
     1021 gtcgtctgcc agttccgcgt gcgaaaatcc attccctgga ctacaaagac catgacggtg
     1081 attataaaga tcatgacatc gattacaagg atgacgatga caagatggac atcgcggatc
     1141 tgcgtaccct gggttacagc cagcagcagc aggagaagat caagccgaag gtgcgcagca
     1201 ccgtggctca gcaccacgaa gccctggtgg gccacggttt cacccacgct cacatcgtgg
     1261 ccctgagcca gcacccagcc gcgctgggca ccgtggccgt gaaatatcag gacatgattg
     1321 ctgccctgcc agaggccacc catgaagcta ttgtgggcgt gggcaagcag tggagcggtg
     1381 ctcgtgcgct ggaggcgctg ctgaccgtgg ctggtgaact gcgtggtccg ccgctgcagc
     1441 tggataccgg tcagctgctg aaaattgcca aacgtggcgg tgtgaccgcg gtggaggccg
     1501 tccacgcctg gaggaatgct ctcacaggcg ctcccttgaa cctgacccct cagcaagtgg
     1561 tggcaattgc atctaatatt gggggtaaac aggctcttga gactgtgcag agactgcttc
     1621 ccgtgctctg tcaggcccac ggactcacac cagaacaggt tgtcgccatc gcatccaacg
     1681 gaggaggtaa gcaagccctg gaaacagtgc agcgtttgct cccggtgctg tgccaggcgc
     1741 atggattaac accggagcag gtcgtggcta tcgcctctaa cggcgggggg aaacaggcct
     1801 tagagacggt acaaaggttg ctgcccgtgc tatgccaggc tcacgggctc actccgcaac
     1861 aggtcgttgc tatcgctagc aacatcgggg ggaagcaggc cctcgaaact gtccagaggc
     1921 tgctgcctgt gttgtgtcaa gcccatggat tgacacccca gcaagtagtc gccatagcct
     1981 ccaacatagg aggcaagcag gcgctggaga ccgtacagcg gctcttgccc gtgctttgtc
     2041 aggctcatgg gctgactcct caacaggtgg tggctattgc ctccaatatc ggagggaaac
     2101 aagcgcttga aaccgtccaa agactcttgc ctgttctgtg tcaggcacac gggctgacgc
     2161 ctgaacaagt ggtagcaatt gcttctaata acggcggaaa gcaggcactg gaaaccgtgc
     2221 aagcccttct tccagtatta tgccaggcac atggtctgac acctgagcag gtggtagcta
     2281 tcgcttcaaa caatggtggc aaacaggcac ttgaaactgt gcaggcactg ttacccgtcc
     2341 tatgccaagc acacggtctg acccccgagc aagtagtggc catcgcctca aatggaggcg
     2401 ggaagcaagc acttgagacc gtgcagaggc tcttgccagt cttatgtcag gcgcacggtc
     2461 taacccctga gcaagtggtc gctatagctt caaacggggg cggcaagcaa gcattagaga
     2521 ctgtacagcg gttgttgcct gtgctttgcc aagctcacgg actgacccct gaacaggtcg
     2581 tcgcgatcgc ttcacatgat ggcggaaaac aagctctaga gaccgttcag gccctgctgc
     2641 cggtcctctg ccaggcccat ggcttgacgc cagagcaggt agttgccatc gcaagcaaca
     2701 atgggggaaa acaggccctg gagacggttc aggcgctact accagtactc tgtcaagcac
     2761 atgggcttac tcccgaacaa gttgttgcaa tcgcgagcaa tggagggggt aagcaggcct
     2821 tggagacagt tcaacggctt ctccctgtac tctgccaagc ccacggctta actcccgagc
     2881 aggttgtagc catagccagt aatggcggtg gaaagcaagc gttggaaaca gttcagcgat
     2941 tgcttccggt tctatgtcag gcccacggcc tgacgcccga acaggtggtc gcaatcgcat
     3001 ctaatggggg aggtaaacaa gctttagaaa ccgttcagcg gctcctgccg gtgttgtgcc
     3061 aagctcatgg gttgacccca gagcaagtcg tcgctattgc cagcaacaac ggcggcaaac
     3121 aagcactcga gacggtgcaa gctctactgc ccgtcctgtg tcaagcgcac ggactgaccc
     3181 cacagcaagt tgtggccatt gcgtcaaacg gtggcggcag gcctgcgctg gaatccatcg
     3241 ttgcccagct gtctagacct gacccggccc tagccgcact taccaacgat cacctggtgg
     3301 cgctggcttg cctgggcggt cgtccggccc tggatgccgt gaagaaaggc ctgggtggat
     3361 ccggcagcta cgccctgggt ccgtatcaga ttagcgcccc gcagctgcca gcttacaatg
     3421 gtcagaccgt gggtaccttc tactatgtga acgacgcggg cggtctggag agcaaggtgt
     3481 ttatcagcgg cggtccaacc ccgtacccaa actatgtcag tgccggtcat gtggccggtc
     3541 agagcgccct gttcatgcgt gataacggca tcagcgaggg tctggtgttc cacaacaacc
     3601 cgaaaggcac ctgcggtttt tgcgtgaaca tgatcgagac cctgctgccg gaaaacgcga
     3661 aaatgaccgt ggtgccgccg gaaggttctg gcggctcaac taatctgagc gacatcattg
     3721 agaaggagac tgggaaacag ctggtcattc aggagtccat cctgatgctg cctgaggagg
     3781 tggaggaagt gatcggcaac aagccagagt ctgacatcct ggtgcacacc gcctacgacg
     3841 agtccacaga tgagaatgtg atgctgctga cctctgacgc ccccgagtat aagccttggg
     3901 ccctggtcat ccaggattct aacggcgaga ataagatcaa gatgctggga agcggagcta
     3961 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctatggtgt
     4021 ccaagggcga ggaagataac atggccatca tcaaggagtt catgaggttt aaggtccaca
     4081 tggagggttc agtcaatggc cacgagttcg agattgaagg cgagggcgag ggccgcccct
     4141 acgaagggac acagacggcg aaattgaagg tgaccaaagg cgggccattg cccttcgcat
     4201 gggacatctt gtcccctcag tttatgtatg gcagcaaggc ctatgttaag caccccgctg
     4261 atatcccgga ctacttgaag ctgtcctttc cagaggggtt taaatgggag cgcgttatga
     4321 atttcgaaga cggaggagtg gttacggtga cgcaggactc atccctgcag gacggagaat
     4381 ttatatataa ggttaagttg agaggcacaa acttcccaag cgacggccct gtgatgcaga
     4441 agaaaacaat ggggtgggaa gcttccagcg agcgcatgta ccccgaagat ggcgccctca
     4501 agggcgagat aaagcaaagg ctgaaactta aggacggcgg tcattacgac gcggaggtca
     4561 agacaactta caaggctaaa aaacccgttc agttgcctgg ggcttacaat gttaatatca
     4621 aacttgacat cacaagccac aatgaagact atacgatcgt ggagcagtat gaacgagcgg
     4681 aaggcaggca ctcaacgggg gggatggacg agctttacaa gtgataaggg gtctttgtcc
     4741 tctgtactgt ctctctcctt gcccctaacc caaaaagctt catttttctg tgtaggctgc
     4801 acaagagcct tgattgaaga tatattcttt ctgaacagta tttaaggttt ccaataaaat
     4861 gtacacccct cagaacttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     4921 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4981 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5041 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5101 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5161 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5221 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5281 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5341 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     5401 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     5461 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     5521 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     5581 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     5641 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     5701 gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca
     5761 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagcctttg
     5821 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     5881 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     5941 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     6001 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     6061 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     6121 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     6181 tctggttatg tgtgggaggg ctaagcactt cgtggccgag gagcaggact gacacgtgct
     6241 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     6301 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     6361 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6421 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6481 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     6541 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6601 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6661 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6721 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6781 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6841 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6901 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6961 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7021 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7081 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7141 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7201 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7261 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7321 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7381 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7441 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7501 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7561 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7621 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7681 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7741 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7801 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7861 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7921 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7981 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8041 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8101 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8161 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8221 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8281 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8341 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8401 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8461 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8521 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8581 catttatcag ggttattgtc tcatgagcgg atacatattt gaat
//
