LOCUS       sequence-360540-        4750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian cell plasmid encoding pH-Countdown targeted to 
            mitochondria via fusion to the COX8A presequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4750)
  AUTHORS   Seidel ZP, Wang JCK, Riegler J, York AG, Ingaramo M
  TITLE     Relaxation Sensors
  JOURNAL   (2021), doi: 10.5281/zenodo.5810930
REFERENCE   2  (bases 1 to 4750)
  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: "(2021), 
            doi: 10.5281/zenodo.5810930"
COMMENT     Sequence Label: N1-Mito-pHCountdown
FEATURES             Location/Qualifiers
     source          1..4750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             651..725
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     polyA_signal    1592..1713
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1629..1648)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1683..1702
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1720..2175)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1857..1878)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2069..2088
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2202..2306
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2308..2665
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2308..2499
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2331..2351)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2516..2651
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2700..3494
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2700..3494
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2754..2773)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3364..3383
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3682..3701)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3726..3773
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4102..4690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4591..4610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc atgtccgtcc
      661 tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca gtgccgcgcg
      721 ccaagatcca ttcgttgggg gatccaccgg taatggtgag caagggcgag gagaacaaca
      781 tggccgtgat taaaccagac atgaagatca agctgcgtat ggaaggctct gtaaatggac
      841 accgtttcag aattgaagga gttggccttg ggaagcctct tgagggaaaa cagagtatgg
      901 accttaaagt caaagaaggc ggacctctgc ctttcgccta tgacatcttg acaatggcgt
      961 tctgttacgg taacagggta ttcgccaaat acccagaaaa tatagtagac tatttcaagc
     1021 agtcgtttcc tgagggctac tcttgggaac gacaaatgat ttacgaagac gggggcattt
     1081 gtgtcgcgac aaacgacata accctggatg gtgactgtat gatctccgaa attcgattta
     1141 agggtgtgaa ctttcctgcc aatggtccag ttttccagaa gaggactgtg aaatgggagc
     1201 tatcccacga gaaattgtat gctcgtgatg gactgctgta ctctgatggt aactacgctc
     1261 tgtcgcttga aggaggtggc cattaccgat gtgacaataa aactacttat aaagctaaga
     1321 aggttgtcca gttgccagac tatcactggg tgacccacag tattgtgatt aaaagccacg
     1381 acaaagatta cagtaatgtt aatctgcatg agcatgccga agcacattct gagctgccga
     1441 ggcaggccat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     1501 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1561 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1621 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1681 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     1741 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1801 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1861 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1921 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     1981 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2041 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2101 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2161 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2221 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2281 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     2341 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2401 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2461 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2521 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2581 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2641 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     2701 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2761 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     2821 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     2881 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     2941 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3001 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3061 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3121 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3181 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     3241 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3301 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3361 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3421 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3481 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     3541 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     3601 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     3661 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     3721 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     3781 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     3841 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     3901 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     3961 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4021 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4081 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4141 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4201 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4261 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4321 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4381 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4441 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4501 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4561 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4621 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4681 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4741 tgctcacatg
//
