LOCUS       sequence-415644-        8318 bp    DNA     circular SYN 15-APR-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 8318)
  TITLE     Quantifying constraint in the human mitochondrial genome
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8318)
  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: 
            "Unpublished"
COMMENT     Sequence Label: m.3075-Left TALE-G1397-N-dead-mCherry
FEATURES             Location/Qualifiers
     source          1..8318
                     /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             3393..3641
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3651..3707
                     /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      4615..5043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4702..4721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4912..4933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5052..5072)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5057..5386
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5237..5372
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5299..5318
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5434..5481
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5500..5898
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6056..6177
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6093..6112)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6147..6166
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6226..6242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6226..6242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6239..6261)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6250..6266
                     /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(6274..6280,6281..6298,6299..6304))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6319..6340
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6457..6474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6628..7216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6708..6727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7387..8178,8179..8247))
                     /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     8010..8029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8248..8318,1..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 tacatgcctg gcgtaacgcg ctgaccggcg ctcctttaaa cctcactcca gagcaggtgg
     1561 ttgccattgc cagtcacgat ggagggaagc aggccctgga gaccgtacag gcactactcc
     1621 cagttttgtg tcaagctcac ggattgaccc ccgaacaggt cgtggccatc gccagccacg
     1681 atgggggaaa acaggcctta gagacagtcc aagcactgct gcccgtgttg tgccaagcac
     1741 acggactgac accggaacag gttgttgcca tagccagcaa tgggggagga aaacaagccc
     1801 tcgaaactgt gcagcgcttg ctccccgtcc tgtgtcaggc ccatggactg acccctcagc
     1861 aggtggtggc tatcgcaagt aacattggag gaaagcaggc tttggaaacc gtacagagat
     1921 tgttgccagt tctgtgccag gctcatggtc taacacctga acaggtagtt gcaatcgcct
     1981 cccatgatgg tggcaagcaa gctttagaaa ccgtgcaggc cctgctgcct gtcttgtgtc
     2041 aggcacacgg gttgacacca gaacaggtgg tcgccattgc gagcaataac gggggcaaac
     2101 aagcacttga aaccgtccaa gccttgcttc cagtactctg ccaggcacat ggacttacgc
     2161 ctgagcaagt ggtggccatt gcatctaatg gtggcgggaa acaggcgctg gaaacagtcc
     2221 aacgactgct gccagtgctg tgccaagccc acggtcttac tcctgaacaa gtggttgcta
     2281 tcgccagcaa caacggaggc aagcaggcac ttgagactgt acaagcactg ttgcccgtat
     2341 tatgccaggc ccacggcctg actcctcaac aggtcgtcgc gatcgctagt aatatcggcg
     2401 gtaagcaggc tctggaaact gttcagagac tcctgcctgt tctatgccag gcgcatgggc
     2461 tgactcccga acaagtagta gccatagcgt ccaacggtgg aggcaaacag gccctcgaga
     2521 ctgtccagcg cctattacct gtgctttgcc aagcgcacgg gttaacgccc gagcaagttg
     2581 tagcgatcgc ctcacacgac ggaggtaaac aagctcttga gacagttcag gctctgcttc
     2641 cagtgctctg tcaagcccat ggtttaaccc cagagcaagt cgtcgctatc gcctctaacg
     2701 ggggtggtaa acaggctctc gagacggtac agagactgct ccctgttctt tgtcaggctc
     2761 acggactcac ccctgagcag gttgtagcaa ttgcaagtaa taacggtgga aaacaggctt
     2821 tagaaacggt tcaggcatta ttacccgtcc tatgtcaagc gcatgggttg acgccacagc
     2881 aggttgtggc tattgccagc aacatagggg gccgaccagc tctggagagt atagtggccc
     2941 aactgtccag accagatccc gcgttggccg ctcttaccaa cgatcacctg gtggcgctgg
     3001 cttgcctggg cggtcgtccg gccctggatg ccgtgaagaa aggcctgggt ggatccggca
     3061 gctacgccct gggtccgtat cagattagcg ccccgcagct gccagcttac aatggtcaga
     3121 ccgtgggtac cttctactat gtgaacgacg cgggcggtct ggagagcaag gtgtttatca
     3181 gcggcggtcc aaccccgtac ccaaactatg tcagtgccgg tcatgtggcc ggtcagagcg
     3241 ccctgttcat gcgtgataac ggcatcagcg agggtctggt gttccacaac aacccgaaag
     3301 gcacctgcgg tttttgcgtg aacatgatcg agaccctgct gccggaaaac gcgaaaatga
     3361 ccgtggtgcc gccggaaggt tctggcggct caactaatct gagcgacatc attgagaagg
     3421 agactgggaa acagctggtc attcaggagt ccatcctgat gctgcctgag gaggtggagg
     3481 aagtgatcgg caacaagcca gagtctgaca tcctggtgca caccgcctac gacgagtcca
     3541 cagatgagaa tgtgatgctg ctgacctctg acgcccccga gtataagcct tgggccctgg
     3601 tcatccagga ttctaacggc gagaataaga tcaagatgct gggaagcgga gctactaact
     3661 tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg gtgtccaagg
     3721 gcgaggaaga taacatggcc atcatcaagg agttcatgag gtttaaggtc cacatggagg
     3781 gttcagtcaa tggccacgag ttcgagattg aaggcgaggg cgagggccgc ccctacgaag
     3841 ggacacagac ggcgaaattg aaggtgacca aaggcgggcc attgcccttc gcatgggaca
     3901 tcttgtcccc tcagtttatg tatggcagca aggcctatgt taagcacccc gctgatatcc
     3961 cggactactt gaagctgtcc tttccagagg ggtttaaatg ggagcgcgtt atgaatttcg
     4021 aagacggagg agtggttacg gtgacgcagg actcatccct gcaggacgga gaatttatat
     4081 ataaggttaa gttgagaggc acaaacttcc caagcgacgg ccctgtgatg cagaagaaaa
     4141 caatggggtg ggaagcttcc agcgagcgca tgtaccccga agatggcgcc ctcaagggcg
     4201 agataaagca aaggctgaaa cttaaggacg gcggtcatta cgacgcggag gtcaagacaa
     4261 cttacaaggc taaaaaaccc gttcagttgc ctggggctta caatgttaat atcaaacttg
     4321 acatcacaag ccacaatgaa gactatacga tcgtggagca gtatgaacga gcggaaggca
     4381 ggcactcaac gggggggatg gacgagcttt acaagtgata aggggtcttt gtcctctgta
     4441 ctgtctctct ccttgcccct aacccaaaaa gcttcatttt tctgtgtagg ctgcacaaga
     4501 gccttgattg aagatatatt ctttctgaac agtatttaag gtttccaata aaatgtacac
     4561 ccctcagaac ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     4621 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4681 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4741 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4801 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4861 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4921 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4981 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5041 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     5101 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     5161 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     5221 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5281 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     5341 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     5401 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
     5461 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagcc tttgtctcaa
     5521 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     5581 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     5641 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     5701 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     5761 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     5821 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     5881 tatgtgtggg agggctaagc acttcgtggc cgaggagcag gactgacacg tgctacgaga
     5941 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6001 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     6061 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6121 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6181 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     6241 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     6301 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     6361 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6421 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     6481 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     6541 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     6601 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6661 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6721 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6781 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6841 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6901 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6961 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     7021 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     7081 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     7141 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     7201 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     7261 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7321 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7381 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7441 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7501 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7561 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7621 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7681 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7741 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7801 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7861 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7921 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7981 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     8041 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     8101 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     8161 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     8221 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     8281 tcagggttat tgtctcatga gcggatacat atttgaat
//
