LOCUS       sequence-422728-        6673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial protein expression of Pacer RH domain mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6673)
  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 6673)
  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: 2CT-PacerRH R628K
FEATURES             Location/Qualifiers
     source          1..6673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2082..2101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2242..2264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3907..3926
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4010..4029
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4010..4028
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4186..5286
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     5260..5283
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5341..5361
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6209..6227)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6223..6270
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6636..6641,6642..6658,6659..6664))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
     3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa
     4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat
     4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa
     4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
     4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
     4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
     4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
     4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
     4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
     4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
     4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac
     4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
     4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
     4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
     4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
     4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
     4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
     5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
     5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc
     5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     5281 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag
     5341 gaaaacctgt acttccaatc caatgcaagg gacctggtgg tggccgctca gaacttcttc
     5401 tgcgctggtt gtggaacccc tgtggagcct aagttcgtga agagactgcg ttactgcgaa
     5461 tacctgggaa agtacttctg cgactgctgc cactcctacg ctgagtcttg catcccagcc
     5521 cgcatcctga tgatgtggga cttcaagaag tactacgtgt ccaacttctc taagcagctg
     5581 ctggacagca tctggcacca gcccatcttc aacctgctga gcatcggaca gtccctgtac
     5641 gccaaggcta aggaactgga cagagtgaag gagatccagg aacagctgtt ccacatcaag
     5701 aagctgctga agacctgccg tttcgccaac tctgctctga aggagttcga gcaggtgcct
     5761 ggccacctga ccgacgagct gcacctgttc agcctggaag acctggtgag aatcaagaag
     5821 ggcctgctgg ctccactgct gaaggacatc ctgaaggcca gcctggctca cgtggctgga
     5881 tgtgagctgt gccagggcaa gggcttcatc tgcgagttct gccagaacac caccgtgatc
     5941 ttccccttcc agactgctac ctgccgccgc tgctccgcct gcaaagcttg cttccacaag
     6001 cagtgcttcc agagcagcga gtgccctaga tgtgcccgta ttactgctcg tagaaaactc
     6061 ctggaaagtg tcgcctccgc cgccaccatt ggaagtggat aacggatccg cgatcgcggc
     6121 gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc
     6181 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     6241 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca
     6301 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag
     6361 caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa
     6421 ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg
     6481 gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     6541 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     6601 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     6661 tcaaacatga gaa
//
