LOCUS       sequence-500890-        7198 bp    DNA     circular SYN 23-FEB-2026
DEFINITION  For biolistic transformation to express Arg8m* and a non-targeting 
            control sgRNA from mtDNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7198)
  AUTHORS   Yin S, Jarosz DF, Ting AY
  TITLE     Towards CRISPR-based editing of the mitochondrial genome in yeast.
  JOURNAL   Proc Natl Acad Sci U S A. 2026 Jan 20;123(3):e2505894123. doi: 
            10.1073/pnas.2505894123. Epub 2026 Jan 16.
  PUBMED    41543903
REFERENCE   2  (bases 1 to 7198)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 23, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2026-01-20"; volume: "123(3):e2505894123. 
            doi"; pages: " 10.1073/pnas.2505894123. Epub 2026 Jan 16"
COMMENT     Sequence Label: pPT24-mtArg8m*-V5-UTR-HH-ntsgRNA-HDV
FEATURES             Location/Qualifiers
     source          1..7198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2217..2233
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2218..2234
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3866..3907
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     ncRNA           4048..4084
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        4112..4186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           4195..4262
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(6509..6525)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6509..6525)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6557..6576)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6558..6576)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6583..6600)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6583..6599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6592..6614)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6741..7196
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6828..6847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7038..7059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagctcga aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga
     2221 ggtcgacggt atcgataagc ttatcggcgg ggaccccgaa ggagtataaa taaaaattaa
     2281 taatatatta tatatatatt atattaataa taataataat aataataaat aataactcct
     2341 tgcttcatac ctttataaat aaggtaatca ctaatatatt ataataataa taattatatt
     2401 tataatttat atatttatat atataaaaat tatatatatt atatataatc taaatattat
     2461 atattttaat aaatattaat atatatgata tgaatattat tagttttcgg gaagcgggaa
     2521 ttcataaatt ttaattaaaa gtagtattaa catattataa atagacaaaa gagtctaaag
     2581 gttaagattt attcatatgt tcaaaagata tttatcatca acatcatcaa gaagattcac
     2641 atcaatctta gaagaaaaag cttttcaagt aacaacatat tcaagacctg aagatttatg
     2701 tatcacaaga ggtaaaaatg ctaaattata tgatgatgta aatggtaaag aatatatcga
     2761 tttcacagct ggtattgctg taacagcttt aggtcatgct aatcctaaag tagccgaaat
     2821 cttacatcac taagctaata aattagtcca ttcatcaaat ttatatttca caaaagaatg
     2881 tttagattta tcagaaaaaa tcgtagaaaa aacaaaacaa ttcggtggtc aacatgatgc
     2941 ttcaagagta ttcttatgta attcaggtac cgaagctaat gaagctgcat taaaattcgc
     3001 taaaaaacat ggtatcatga aaaatcctag taaacaaggt attgtagctt tcgaaaattc
     3061 attccatggt agaacaatgg gtgctttatc agtaacatga aattcaaaat atagaacacc
     3121 tttcggtgat ttagtacctc atgtatcatt cttaaattta aatgatgaaa tgacaaaatt
     3181 acaatcatat atcgaaacaa aaaaagatga aatcgctggt ttaatcgtag aacctattca
     3241 aggtgaaggt ggtgtatttc ctgtagaagt agaaaaatta actggtttaa aaaaaatctg
     3301 tcaagataat gatgtaatcg taattcatga tgaaattcaa tgtggtttag gtagatcagg
     3361 taaattatga gctcatgctt atttaccttc agaagctcat cctgatattt tcacatcagc
     3421 taaagcatta ggtaatggtt tccctattgc tgctacaatc gtaaatgaaa aagtaaataa
     3481 tgctttaaga gtaggtgatc atggtacaac atatggtggt aatcctttag cttgttcagt
     3541 atcaaattat gtattagata caattgctga tgaagcattc ttaaaacaag tatcaaaaaa
     3601 atcagatatc ttacaaaaaa gattgagaga aatccaagct aaatatccta atcaaatcaa
     3661 aacaatcaga ggtaaaggtt taatgttagg tgctgaattt gtagaacctc caacagaagt
     3721 aatcaaaaaa gctagagaat taggtttatt aatcatcaca gctggtaaat caacagtaag
     3781 attcgtacct gctttaacaa tcgaagatga attaatcgaa gaaggtatgg atgctttcga
     3841 aaaagctatc gaagctgtat atgctggcaa gcccatcccc aaccccttgt taggcttaga
     3901 cagcacctaa ctcgagttaa tatttactta ttattaatat ttttaattat taaaaataat
     3961 aataataata ataattataa taatattctt aaatataata aagatataga tttatattct
     4021 attcaatcac cttatgaatt cagcttactg atgagtccgt gaggacgaaa cgagtaagct
     4081 cgtcctagat gcgagatatc ctcatgccta ggttttagag ctagaaatag caagttaaaa
     4141 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc ttttggccgg
     4201 catggtccca gcctcctcgc tggcgccggc tgggcaacat gcttcggcat ggcgaatggg
     4261 acgaattccg taaggagtga gggacccctc cctatactaa cgggaggggg accgaacccc
     4321 gaaggagttt tatttttagt attttataaa atatatattt atatgattaa taatattata
     4381 tatattattt ataaaaataa tatataattt taattatttt taataaaaaa aggtggggtt
     4441 tggtaatata atatttttat tttatttata atatataata ataaattata aataaatttt
     4501 aattaaaagt agtattaaca tattataaat agacaaaaga gtctaaaggt taagatttat
     4561 taaaatgtta gatttattaa gattacaatt aacaacattc attatgaatg atgtaccaac
     4621 accttatgca tgttattttc aggattcagc aacaccaaat caagaaggta ttttagaatt
     4681 acatgataat attatgtttt atttattagt tattttaggt ttagtatctt gaatgttata
     4741 tacaattgtt ataacatatt caaaaaatcc tattgcatat aaatatatta aacatggaca
     4801 aactattgaa gttatttgaa caatttttcc agctgtaatt ttattaatta ttgctttccc
     4861 ttcatttatt ttattatatt tatgtgatga agttatttca ccagctataa ctattaaagc
     4921 tattggatat caatgatatt gaaaatatga atattcagat tttattaatg atagtggtaa
     4981 actgttgaat tttgaatcat atgttattcc tgatgaatta ttagaagaag gacaattaag
     5041 attattagat actgatactt ctatagttgt acctgtagat acacatatta gattcgttgt
     5101 aacagctgct gatgttattc atgattttgc tatcccaagt ttaggtatta aagttgatgc
     5161 tactcctggt agattaaatc aagtttctgc tttaattcaa agagaaggtg tcttctatgg
     5221 ggcatgttct gagttgtgtg ggacaggtca tgcaaatatg ccaattaaga tcgaagcagt
     5281 atcattacct aaatttttgg aatgattaaa ttaacaataa ttaatattta cttattatta
     5341 atatttttaa ttattaaaaa taataataat aataataatt ataataatat tcttaaatat
     5401 aataaagata tagatttata ttctattcaa tcaccttata ttaaaaatat aaatattatt
     5461 aaaagaggtt atcatacttc tttaaataat aaattaatta ttgttcaaaa agataataaa
     5521 aataataata agaataattt agaaatagat aatttttata aatgattagt aggatttaca
     5581 gatggagatg gtagttttta tattaaatta aatgataaaa aatatttaag atttttttat
     5641 ggttttagaa tacatattga tgataaagca tgtttagaaa agattagaaa tatattaaat
     5701 ataccttcta attttgaaga actacttaaa acaattatat tagtaaattc acaaaagaaa
     5761 tggttatatt ctaatattgt aactattttt gataagtatc cttgtttaac aattaaatat
     5821 tatagttatt ataaatgaaa aatagctata attaataatt taaatggtat atcttataat
     5881 aataaagatt tattaaatat taaaaataca attaataatt atgaagttat acctaattta
     5941 aaaattccat atgataaaat aaatgattat tgaattttag gttttattga agctgaaggt
     6001 tcatttgatc tatctccaaa acgtaatatt tgtggtttta atgtttcaca acataaacgt
     6061 agtattaata cattaaaagc tattaaatct tatgtattaa ataattgaaa accaattgat
     6121 aatacaccat tattaattaa aaataaatta ttaaaagatt gagattcatc tattaaatta
     6181 actaaacctg ataaaaatgg agttattaaa ttagaattta atagaataga ttttttatat
     6241 tatgttattt tacctaaatt atattcatta aaatgatata gtcgtaaaga aattgatttc
     6301 caattatgaa aaacacttat agaaatctat ataaaaggtt tacataatac acttaaaggt
     6361 tctaatttat taaaattaat taataataat attaataaaa aaagatatta ttctaattat
     6421 aatatttctc ctttcggggt tccggctccc gtggccgggc cccgatgata agctgtcaaa
     6481 catgagaatt aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg
     6541 gtggagctcc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     6601 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6661 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6721 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6781 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     6841 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6901 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     6961 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     7021 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     7081 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     7141 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttag
//
