LOCUS       sequence-489309-        8530 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  A piggyBac vector containing the NDUFB9 coding sequence (CDS) 
            flanked by the 5' and 3' UTRs of the Actb gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8530)
  AUTHORS   Luo J, Khandwala S, Hu J, Lee SY, Hickey KL, Levine ZG, Harper JW, 
            Ting AY, Weissman JS
  TITLE     Proximity-specific ribosome profiling reveals the logic of localized
            mitochondrial translation.
  JOURNAL   Cell. 2025 Aug 25:S0092-8674(25)00916-X. doi: 
            10.1016/j.cell.2025.08.002.
  PUBMED    40876456
REFERENCE   2  (bases 1 to 8530)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Aug 25:S0092-8674(25)00916-X. doi: 10.1016/j.cell.2025.08.002."
COMMENT     Sequence Label: pJL306
FEATURES             Location/Qualifiers
     source          1..8530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_feature    1403..1991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1509..1520
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1918..1938
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2016..2615)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAS"
     primer_bind     complement(2102..2122)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(2616..2669)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2679..3386)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2954..2973)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3223..3241
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     promoter        complement(3398..3898)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3461..3479)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     promoter        3985..5163
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4216..5154
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5111..5131
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   5984..6018
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6509..6525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6509..6525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6522..6544)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6533..6549
                     /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(6557..6563,6564..6581,6582..6587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6602..6623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6740..6757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6911..7499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6991..7010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7670..8461,8462..8530))
                     /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     8293..8312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc agggtttatt acagggacag cagagatcca
     1381 gtttggttaa ttagctagcg atgcggggag gcggcccaaa gggagatccg actcgtctga
     1441 gggcgaaggc gaagacgcgg aagaggccgc agagccggca gcaggccgcg ggaaggaagg
     1501 tccgctggat tgagggccga agggacgtag cagaaggacg tcccgcgcag aatccaggtg
     1561 gcaacacagg cgagcagcca aggaaaggac gatgatttcc ccgacaacac cacggaattg
     1621 tcagtgccca acagccgagc ccctgtccag cagcgggcaa ggcaggcggc gatgagttcc
     1681 gccgtggcaa tagggagggg gaaagcgaaa gtcccggaaa ggagctgaca ggtggtggca
     1741 atgccccaac cagtgggggt tgcgtcagca aacacagtgc acaccacgcc acgttgcctg
     1801 acaacgggcc acaactcctc ataaagagac agcaaccagg atttatacaa ggaggagaaa
     1861 atgaaagcca tacgggaagc aatagcatga tacaaaggca ttaaagcagc gtatccacat
     1921 agcgtaaaag gagcaacata gttaagaata ccagtcaatc tttcacaaat tttgtaatcc
     1981 agaggttgat taccgataag cttgatatcg aattcttaac tggcaccggg cttgcgggtc
     2041 atgcaccagg tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc
     2101 cgctcgtaga aggggaggtt gcggggcgcg gaggtctcca ggaaggcggg caccccggcg
     2161 cgctcggccg cctccactcc ggggagcacg acggcgctgc ccagaccctt gccctggtgg
     2221 tcgggcgaga cgccgacggt ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc
     2281 aggaggcctt ccatctgttg ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc
     2341 gggccgatct cggcgaacac cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc
     2401 acggcggcgc cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg
     2461 aagagttctt gcagctcggt gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc
     2521 gtggcggggt agtcggcgaa cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg
     2581 cgggtggcga ggcgcaccgt gggcttgtac tcggttgggc caggattctc ctcgacgtca
     2641 ccgcatgtta gcagacttcc tctgccctct ccactgccct tgtacagctc gtccatgccg
     2701 ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg
     2761 tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc
     2821 ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc
     2881 ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc ggaggaggcc
     2941 tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc
     3001 agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc
     3061 acgccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg gaaggacagc
     3121 ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac
     3181 tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac cttcagcttg
     3241 gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg
     3301 ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat gatggccatg
     3361 ttatcctcct cgcccttgct caccatggtg gtaccccctg gggagagagg tcggtgattc
     3421 ggtcaacgag ggagccgact gccgacgtgc gctccggagg cttgcagaat gcggaacacc
     3481 gcgcgggcag gaacagggcc cacactaccg ccccacaccc cgcctcccgc accgcccctt
     3541 cccggccgct gctctcggcg cgccctgctg agcagccgct attggccaca gcccatcgcg
     3601 gtcggcgcgc tgccattgct ccctggcgct gtccgtctgc gagggtacta gtgagacgtg
     3661 cggcttccgt ttgtcacgtc cggcacgccg cgaaccgcaa ggaaccttcc cgacttaggg
     3721 gcggagcagg aagcgtcgcc ggggggccca caagggtagc ggcgaagatc cgggtgacgc
     3781 tgcgaacgga cgtgaagaat gtgcgagacc cagggtcggc gccgctgcgt ttcccggaac
     3841 cacgcccaga gcagccgcgt ccctgcgcaa acccagggct gccttggaaa aggcgcaaat
     3901 cgatcgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
     3961 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     4021 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
     4081 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     4141 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     4201 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
     4261 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
     4321 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     4381 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
     4441 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     4501 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     4561 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     4621 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     4681 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     4741 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     4801 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     4861 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     4921 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     4981 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     5041 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     5101 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     5161 tgagggatcc accgccgaga ccgcgtccgc cccgcgagca cagagcctcg cctttgccga
     5221 tccgccgccc gtccacaccc gccgccagct caccatggcg ttcttggcgt cgggacccta
     5281 cctgacccat cagcaaaagg tgttgcggct ttataagcgg gcgctacgcc acctcgagtc
     5341 gtggtgcgtc cagagagaca aataccgata ctttgcttgt ttgatgagag cccggtttga
     5401 agaacataag aatgaaaagg atatggcgaa ggccacccag ctgctgaagg aggccgagga
     5461 agaattctgg taccgtcagc atccacagcc atacatcttc cctgactctc ctgggggcac
     5521 ctcctatgag agatacgatt gctacaaggt cccagaatgg tgcttagatg actggcatcc
     5581 ttctgagaag gcaatgtatc ctgattactt tgccaagaga gaacagtgga agaaactgcg
     5641 gagggaaagc tgggaacgag aggttaagca gctgcaggag gaaacgccac ctggtggtcc
     5701 tttaactgaa gctttgcccc ctgcccgaaa ggaaggtgat ttgcccccac tgtggtggta
     5761 tattgtgacc agaccccggg agcggcccat gtagttaatt aacttttggt ctccctggga
     5821 gtgggtggag gcagccaggg cttacctgta cactgacttg agaccagttg aataaaagtg
     5881 cacaccttaa aaatgagttt aaaccaagcg gcgactgaga tgtcctaaat gcacagcgac
     5941 ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg
     6001 cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg gtcttttttc
     6061 cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc
     6121 ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg ctgctgcatt
     6181 gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc catgcacgcc
     6241 tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg gcttttccgg
     6301 acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac cggcgacagc
     6361 cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg atattacgtc
     6421 agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc ccgtgagtta
     6481 cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     6541 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6601 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6661 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6721 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6781 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6841 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6901 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6961 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     7021 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     7081 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     7141 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7201 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7261 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7321 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7381 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7441 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7501 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7561 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7621 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7681 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7741 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7801 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7861 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7921 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7981 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     8041 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     8101 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     8161 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8221 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8281 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8341 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8401 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8461 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8521 gaatactcat
//
