LOCUS       sequence-401352-        5776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression plasmid of S. cerevisiae Atg32 amino acids 
            200 - 341 in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5776)
  AUTHORS   Xia X, Katzenell S, Reinhart EF, Bauer KM, Pellegrini M, Ragusa MJ
  TITLE     A pseudo-receiver domain in Atg32 is required for mitophagy.
  JOURNAL   Autophagy. 2018;14(9):1620-1628. doi: 10.1080/15548627.2018.1472838.
            Epub 2018 Aug 1.
  PUBMED    29909755
REFERENCE   2  (bases 1 to 5776)
  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: 
            "Autophagy."; date: "2018"; volume: "14(9):1620-1628. doi"; pages: "
            10.1080/15548627.2018.1472838. Epub 2018 Aug 1"
COMMENT     Sequence Label: Atg32 [200-341] 1B
FEATURES             Location/Qualifiers
     source          1..5776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..86
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      197..785
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     686..705
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     939..956
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    971..1110
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1036..1055)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1196..1218
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1212..1403)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2212..3294)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3256..3275
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3295..3372)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3578..3597
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3681..3700
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3681..3699
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3700..3724
                     /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)."
     RBS             3739..3761
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3780..3797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3798..3818
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4320..4337
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4390..4408)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4404..4451
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4488..4943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4575..4594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4785..4806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5036..5776,1..75))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(5036..5776,1..75))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     join(5759..5776,1..2)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
       61 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
      121 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
      181 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      241 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      301 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      361 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      421 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      481 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      541 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      601 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      661 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      721 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      781 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      841 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      901 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      961 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     1021 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     1081 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1141 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1201 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1261 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1321 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1381 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1441 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1501 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1561 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1621 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1681 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1741 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1801 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1861 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1921 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1981 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     2041 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     2101 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     2161 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     2221 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2281 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     2341 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     2401 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     2461 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     2521 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     2581 aacgataccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     2641 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2701 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2761 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2821 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2881 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2941 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     3001 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     3061 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     3121 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     3181 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     3241 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     3301 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     3361 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     3421 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     3481 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3541 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     3601 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     3661 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3721 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggttcttctc
     3781 accatcacca tcaccatgaa aacctgtact tccaatccaa tgcaaccaac agctttgtga
     3841 tgccgaaact gagcctgacc caaaagaacc cggttttccg tctgctgatc ctgggtcgta
     3901 ccggcagcag cttttaccaa agcattccga aggagtatca gagcctgttc gaactgccga
     3961 aataccacga cagcgcgacc ttcccgcaat ataccggtat cgtgatcatt tttcaggagc
     4021 tgcgtgaaat ggtgagcctg ctgaaccgta ttgttcagta cagccaaggc aagccggtta
     4081 tcccgatttg ccagccgggc caagttatcc aggtgaagaa cgttctgaaa agcttcctgc
     4141 gtaacaagct ggtgaaactg ctgtttccgc cggtggttgt gaccaacaaa cgtgacctga
     4201 agaagatgtt ccaacgcctg caagacctga gcctggagta tggtgaagat taataacatt
     4261 ggaagtggat aacggatccg aattcgagcg ccgtcgacaa gcttgcggcc gcactcgagc
     4321 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     4381 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     4441 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     4501 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4561 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4621 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4681 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4741 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4801 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4861 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4921 caaactagta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4981 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     5041 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     5101 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     5161 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     5221 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     5281 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     5341 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     5401 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     5461 accggcgcag gaacactgcc agcgcatcaa caatgttttc acctgaatca ggatattctt
     5521 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     5581 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     5641 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     5701 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     5761 cgcgagccca tttata
//
