LOCUS       sequence-422724-        6687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Pacer mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6687)
  AUTHORS   Tudorica DA, Basak B, Puerta Cordova AS, Khuu G, Rose K, Lazarou M, 
            Holzbaur ELF, Hurley JH
  TITLE     A RAB7A phosphoswitch coordinates Rubicon Homology protein 
            regulation of Parkin-dependent mitophagy.
  JOURNAL   J Cell Biol. 2024 Jul 1;223(7):e202309015. doi: 
            10.1083/jcb.202309015. Epub 2024 May 10.
  PUBMED    38728007
REFERENCE   2  (bases 1 to 6687)
  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: "J Cell 
            Biol."; date: "2024-07-1"; volume: "223(7):e202309015. doi"; pages: 
            " 10.1083/jcb.202309015. Epub 2024 May 10"
COMMENT     Sequence Label: pmCherryC1-Pacer K534N, R623T
FEATURES             Location/Qualifiers
     source          1..6687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /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(758..776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1026..1045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3475..3596
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3512..3531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3566..3585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3603..4058)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3740..3761)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3952..3971
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4085..4189
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4191..4548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4191..4382
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4214..4234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4399..4534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4583..5377
                     /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             4583..5377
                     /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(4637..4656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5247..5266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5565..5584)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5609..5656
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5985..6573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6474..6493
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tccggactca gatctatggt ctcacagtct acagtccgcc aggattcacc agtggagcct
     1381 tgggagggta tttcagatca ttcaggtatc attgacggca gccctagact gctgaacact
     1441 gaccaccctc cctgccagct ggacatccgc ctgatgaggc acaaggccgt gtggatcaac
     1501 cctcaggacg tgcagcagca gccacaggac ctccagtctc aggtgcccgc cgctggaaac
     1561 tccggcaccc acttcgtgac tgatgctgct tctcctagcg gtccctcccc ttcttgcctg
     1621 ggcgacagcc tggctgaaac caccctgtct gaagatacta ctgactctgt gggaagcgcc
     1681 tccccacacg gtagctccga gaagtctagc tccttctccc tgtctagcac cgaagtgcac
     1741 atggtgcgcc ccggatacag ccaccgcgtg agcctgccta cctctccagg tatcctggct
     1801 acctccccat accccgaaac cgacagcgcc ttcttcgagc cttctcacct gacctccgcc
     1861 gctgacgagg gtgctgtgca ggtgagccgc aggaccatct cctctaactc tttcagccca
     1921 gaagtgttcg tgctgcccgt ggacgtggag aaggaaaacg ctcacttcta cgtggccgac
     1981 atgatcatca gcgccatgga gaagatgaag tgcaacatcc tgtcccagca gcagaccgag
     2041 tcctggtcta aggaagtgtc tggcctgctg ggaagcgacc agcctgactc cgagatgacc
     2101 ttcgacacca acatcaagca ggaatctgga agctccacct ctagctactc tggttacgag
     2161 ggttgtgccg tgctccaggt gagcccagtg accgaaacca gaacctacca cgacgtgaag
     2221 gaaatctgca agtgcgacgt ggacgagttc gtgatcctgg aactgggtga cttcaacgac
     2281 atcaccgaaa cctgctcctg ctcctgctcc agcagcaagt ccgtgaccta cgaacccgac
     2341 ttcaactctg ctgagctgct ggccaaggaa ctgtaccgcg tgttccagaa gtgctggatt
     2401 ctgtctgtgg tgaacagcca gctggctggc tctctgagcg ccgccggcag catcgtggtg
     2461 aacgaggaat gcgtgaggaa ggacttcgag tcctctatga acgtggtgca ggaaatcaag
     2521 ttcaagtctc gcatcagggg caccgaggac tgggctccac cccgcttcca gatcatcttc
     2581 aacatccacc ctccactgaa gagggacctg gtggtggccg ctcagaactt cttctgcgct
     2641 ggttgtggaa cccctgtgga gcctaagttc gtgaagagac tgcgttactg cgaatacctg
     2701 ggaaagtact tctgcgactg ctgccactcc tacgctgagt cttgcatccc agcccgcatc
     2761 ctgatgatgt gggacttcaa gaagtactac gtgtccaact tctctaagca gctgctggac
     2821 agcatctggc accagcccat cttcaacctg ctgagcatcg gacagtccct gtacgccaag
     2881 gctaaggaac tggacagagt gaaggagatc caggaacagc tgttccacat caagaacctg
     2941 ctgaagacct gccgtttcgc caactctgct ctgaaggagt tcgagcaggt gcctggccac
     3001 ctgaccgacg agctgcacct gttcagcctg gaagacctgg tgagaatcaa gaagggcctg
     3061 ctggctccac tgctgaagga catcctgaag gccagcctgg ctcacgtggc tggatgtgag
     3121 ctgtgccagg gcaagggctt catctgcgag ttctgccaga acaccaccgt gatcttcccc
     3181 ttccagactg ctacctgccg cacctgctcc gcctgcaaag cttgcttcca caagcagtgc
     3241 ttccagagca gcgagtgccc tagatgtgcc cgtattactg ctcgtagaaa actcctggaa
     3301 agtgtcgcct ccgccgccac ctaggtcgac ggtaccgcgg gcccgggatc caccggatct
     3361 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3421 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3481 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3541 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     3601 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3661 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3721 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3781 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3841 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3901 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3961 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4021 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4081 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4141 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4201 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4261 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4321 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4381 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4441 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4501 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4561 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4621 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4681 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4741 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4801 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4861 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4921 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4981 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5041 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5101 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5161 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5221 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5281 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5341 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5401 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5461 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5521 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5581 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5641 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5701 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5761 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5821 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5881 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5941 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6001 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6061 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6121 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6181 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6241 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6301 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6361 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6421 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6481 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6541 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6601 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6661 gtggataacc gtattaccgc catgcat
//
