LOCUS       sequence-415657-        8522 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 8522)
  TITLE     Quantifying constraint in the human mitochondrial genome
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8522)
  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            "Unpublished"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.5147-Left TALE-G1397-N-dead-mCherry
FEATURES             Location/Qualifiers
     source          1..8522
                     /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             3597..3845
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3855..3911
                     /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      4819..5247
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4906..4925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5116..5137
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5256..5276)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5261..5590
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5441..5576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5503..5522
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5638..5685
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5704..6102
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6260..6381
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6297..6316)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6351..6370
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6430..6446)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6430..6446)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6443..6465)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6454..6470
                     /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(6478..6484,6485..6502,6503..6508))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6523..6544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6661..6678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6832..7420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6912..6931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7591..8382,8383..8451))
                     /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     8214..8233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8452..8522,1..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 gtggaagccg
     1501 tccatgcgtg gcgcaacgca ctgacgggcg ccccactgaa tctgacaccc gagcaggtgg
     1561 tcgccatcgc ctcccacgat ggcggcaaac aggctctgga aacagtgcag gccctgctgc
     1621 ctgtgctgtg ccaggcccac ggcctgaccc ctcagcaggt ggtcgccatc gcctctaaca
     1681 tcggcggaaa gcaggctctg gagaccgtgc agcggctgct gcccgtgctg tgccaggccc
     1741 acggcctgac ccctcagcag gtggtcgcca tcgcctctaa catcggcgga aagcaggctc
     1801 tggagaccgt gcagcggctg ctgcccgtgc tgtgccaggc ccacggcctg acacccgagc
     1861 aggtggtcgc catcgcctcc cacgatggcg gcaaacaggc tctggaaaca gtgcaggccc
     1921 tgctgcctgt gctgtgccag gcccacggcc tgacccctga gcaggtggtg gctatcgcct
     1981 ctaatggcgg aggcaagcag gccctggaaa ccgtgcagag actgctgcct gtgctgtgcc
     2041 aggcccatgg actgacccct gagcaggtgg tggctatcgc ctctaatggc ggaggcaagc
     2101 aggccctgga aaccgtgcag agactgctgc ctgtgctgtg ccaggcccat ggactgaccc
     2161 ctcagcaggt ggtcgccatc gcctctaaca tcggcggaaa gcaggctctg gagaccgtgc
     2221 agcggctgct gcccgtgctg tgccaggccc acggcctgac ccctcagcag gtggtcgcca
     2281 tcgcctctaa catcggcgga aagcaggctc tggagaccgt gcagcggctg ctgcccgtgc
     2341 tgtgccaggc ccacggcctg acccctcagc aggtggtcgc catcgcctct aacatcggcg
     2401 gaaagcaggc tctggagacc gtgcagcggc tgctgcccgt gctgtgccag gcccacggcc
     2461 tgacacccga gcaggtggtc gccatcgcct cccacgatgg cggcaaacag gctctggaaa
     2521 cagtgcaggc cctgctgcct gtgctgtgcc aggcccacgg cctgacccct gagcaggtgg
     2581 tggctatcgc ctctaatggc ggaggcaagc aggccctgga aaccgtgcag agactgctgc
     2641 ctgtgctgtg ccaggcccat ggactgacac ccgagcaggt ggtcgccatc gcctcccacg
     2701 atggcggcaa acaggctctg gaaacagtgc aggccctgct gcctgtgctg tgccaggccc
     2761 acggcctgac acccgagcag gtggtcgcca tcgcctccca cgatggcggc aaacaggctc
     2821 tggaaacagt gcaggccctg ctgcctgtgc tgtgccaggc ccacggcctg acccctcagc
     2881 aggtggtcgc catcgcctct aacatcggcg gaaagcaggc tctggagacc gtgcagcggc
     2941 tgctgcccgt gctgtgccag gcccacggcc tcacacccga gcaagtggtg gccatcgcta
     3001 gcaacaacgg cggcaagcag gctctcgaga cagtccaagc tctgctgcca gtgctgtgcc
     3061 aagctcacgg cctgacacct cagcaggtgg tggccatcgc ctcccacgac ggcgggagac
     3121 ctgccctcga atcaatcgtg gctcagctga gtcgccccga cccggcgctg gcggcactta
     3181 ccaatgatca cctggtggcg ctggcttgcc tgggcggtcg tccggccctg gatgccgtga
     3241 agaaaggcct gggtggatcc ggcagctacg ccctgggtcc gtatcagatt agcgccccgc
     3301 agctgccagc ttacaatggt cagaccgtgg gtaccttcta ctatgtgaac gacgcgggcg
     3361 gtctggagag caaggtgttt atcagcggcg gtccaacccc gtacccaaac tatgtcagtg
     3421 ccggtcatgt ggccggtcag agcgccctgt tcatgcgtga taacggcatc agcgagggtc
     3481 tggtgttcca caacaacccg aaaggcacct gcggtttttg cgtgaacatg atcgagaccc
     3541 tgctgccgga aaacgcgaaa atgaccgtgg tgccgccgga aggttctggc ggctcaacta
     3601 atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag gagtccatcc
     3661 tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct gacatcctgg
     3721 tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc tctgacgccc
     3781 ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat aagatcaaga
     3841 tgctgggaag cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga
     3901 accctggacc tatggtgtcc aagggcgagg aagataacat ggccatcatc aaggagttca
     3961 tgaggtttaa ggtccacatg gagggttcag tcaatggcca cgagttcgag attgaaggcg
     4021 agggcgaggg ccgcccctac gaagggacac agacggcgaa attgaaggtg accaaaggcg
     4081 ggccattgcc cttcgcatgg gacatcttgt cccctcagtt tatgtatggc agcaaggcct
     4141 atgttaagca ccccgctgat atcccggact acttgaagct gtcctttcca gaggggttta
     4201 aatgggagcg cgttatgaat ttcgaagacg gaggagtggt tacggtgacg caggactcat
     4261 ccctgcagga cggagaattt atatataagg ttaagttgag aggcacaaac ttcccaagcg
     4321 acggccctgt gatgcagaag aaaacaatgg ggtgggaagc ttccagcgag cgcatgtacc
     4381 ccgaagatgg cgccctcaag ggcgagataa agcaaaggct gaaacttaag gacggcggtc
     4441 attacgacgc ggaggtcaag acaacttaca aggctaaaaa acccgttcag ttgcctgggg
     4501 cttacaatgt taatatcaaa cttgacatca caagccacaa tgaagactat acgatcgtgg
     4561 agcagtatga acgagcggaa ggcaggcact caacgggggg gatggacgag ctttacaagt
     4621 gataaggggt ctttgtcctc tgtactgtct ctctccttgc ccctaaccca aaaagcttca
     4681 tttttctgtg taggctgcac aagagccttg attgaagata tattctttct gaacagtatt
     4741 taaggtttcc aataaaatgt acacccctca gaacttctga ggcggaaaga accagctggg
     4801 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4861 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4921 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4981 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5041 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5101 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5161 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5221 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     5281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5341 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5401 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5461 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5521 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5581 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
     5641 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     5701 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     5761 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     5821 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     5881 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     5941 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     6001 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     6061 attcgtgaat tgctgccctc tggttatgtg tgggagggct aagcacttcg tggccgagga
     6121 gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6181 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6241 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6301 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6361 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     6421 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6481 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6541 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6601 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6661 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     6721 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6781 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6841 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6901 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6961 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7021 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7081 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7141 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7201 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7261 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7321 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7381 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     7441 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7501 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7561 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7621 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7681 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     7741 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7801 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7861 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7921 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7981 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     8041 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8101 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8161 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8221 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8281 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8341 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8401 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     8461 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     8521 at
//
