LOCUS       sequence-373602-        8315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression of ATG5 and ATG12 protein subunit 
            complexes. Internal References: SMC1178.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8315)
  AUTHORS   Fracchiolla D, Chang C, Hurley JH, Martens S
  TITLE     A PI3K-WIPI2 positive feedback loop allosterically activates LC3 
            lipidation in autophagy.
  JOURNAL   J Cell Biol. 2020 Jul 6;219(7). pii: 151802. doi: 
            10.1083/jcb.201912098.
  PUBMED    32437499
REFERENCE   2  (bases 1 to 8315)
  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: "2020-07-6"; volume: "219(7). pii: 151802. doi"; 
            pages: " 10.1083/jcb.201912098"
COMMENT     Sequence Label: pBIG1b_10xHisTEVATG5_ATG12
FEATURES             Location/Qualifiers
     source          1..8315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             442..1233
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1274..1728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1361..1380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1570..1591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1754..1858
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1859..1927,1928..2719)
                     /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     complement(2077..2096)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2890..3478
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3379..3398
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3632..3649
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  3783..4007
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(4074..4607)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4796..4801,4802..4818,4819..4824))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(5220..5253)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5299..5318)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5343..5391
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5486..5577
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     CDS             5623..5652
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5659..5679
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    6643..6777
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        6933..7024
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     polyA_signal    7619..7753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  8068..8233
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa ctgggaggca gaataaatga tcatatcgtc
      121 aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac
      181 ggtcacactg cttccggtag tcaataaacc ggtaagtagc gtatgcgctc acgcaactgg
      241 tccagaacct tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt
      301 tatgactgtt tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac
      361 gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc
      421 cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc gactcaacta
      481 tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg
      541 tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg
      601 gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact
      661 tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac
      721 gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag
      781 cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc
      841 ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc
      901 tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg
      961 aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt
     1021 tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg
     1081 gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga
     1141 caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg
     1201 aaaggcgaga tcaccaaggt agtcggcaaa taatgtctaa caattcgttc aagccgacgg
     1261 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1501 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1561 ttcttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1621 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1681 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1801 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     1861 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     1921 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1981 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2041 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2101 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2161 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2221 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2281 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2341 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2401 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2461 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2521 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     2581 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2641 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     2701 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     2761 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2821 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2881 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2941 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3001 aactggcttc agcagagcgc agataccaaa tactattctt ctagtgtagc cgtagttagg
     3061 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3121 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3181 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3241 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3301 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3361 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3421 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3481 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3541 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3601 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3661 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagacca
     3721 gccgcgtaac ctggcaaaat cggttacggt tgagtaataa atggatgccc tgcgtaagcg
     3781 ggtgtgggcg gacaataaag tcttaaactg aacaaaatag atctaaacta tgacaataaa
     3841 gtcttaaact agacagaata gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc
     3901 atactggact tttgttatgg ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg
     3961 tcgtattaaa gaggggcgtg gccaagggca tggtaaagac tatattcgcg gcgttgtgac
     4021 aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg
     4081 gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa ctttgtatag
     4141 agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata agcaccaagc
     4201 gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg cctccggtgc
     4261 tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc aaacttgggc
     4321 agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg
     4381 aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg ttgggagtag
     4441 gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat ggatttgact
     4501 tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc taactttgtt
     4561 ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca
     4621 taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga
     4681 ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct
     4741 gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagttt
     4801 acgaaccgaa caggcttatg tcaactgggt ttgtgccttc atccgtttcc acggtgtgcg
     4861 tcacccggca accttgggca gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga
     4921 gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg gccttgctgt tcttctacgg
     4981 caaggtgctg tgcacggatc tgcccttgct tcaggagatc ggtagacctc ggccgtcgcg
     5041 gcgcttgccg gtggtgctga ccccggatga agtggttcgc atcctcggtt ttctggaagg
     5101 cgagcatcgt ttgttcgccc aggactctag ctatagttct agtggttggc tacagctttg
     5161 tttgtactat caacaggttg aactgctgat caacagatcc tctacgcggc cgcggtacca
     5221 taacttcgta tagcatacat tatacgaagt tatctgggtt taaactatat ctcaatcggg
     5281 gtttttatcg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     5341 gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg
     5401 gggttcggtc ccagggctgg cactctgtca agtgaacgct ctatggtcta aagatttaaa
     5461 tcgacctact ccggaatatt aatagatcat ggagataatt aaaatgataa ccatctcgca
     5521 aataaataag tattttactg ttttcgtaac agttttgtaa taaaaaaacc tataaatatt
     5581 ccggattatt cataccgtcc caccatcggg cgcggatcca tgcaccatca ccatcaccat
     5641 caccatcacc atggcggtga aaacctgtat ttccagggct ccggtatgac ggatgataaa
     5701 gatgttctga gagatgtctg gttcggacgc attcctacct gcttcacgct gtaccaagat
     5761 gagattacgg agagggaggc tgaaccctac tacctgctgc tgccaagagt cagctacctg
     5821 actctggtga ccgacaaggt caagaagcac ttccagaagg tcatgaggca ggaggacatc
     5881 tctgaaatct ggttcgagta cgaaggaact cctctgaagt ggcactaccc catcggtctg
     5941 ctgttcgacc tgctggcttc cagctctgcc ctgccttgga acatcaccgt ccacttcaag
     6001 agcttcccag agaaggacct gctgcactgc ccttcaaagg acgctatcga ggcccacttc
     6061 atgtcctgca tgaaggaagc tgacgccctg aagcacaagt cccaggtcat caacgaaatg
     6121 cagaagaagg accacaagca gctgtggatg ggtctgcaaa acgaccgctt cgaccagttc
     6181 tgggctatca accgtaagct gatggagtac cctgccgagg aaaacggctt ccgctacatc
     6241 cccttccgta tctaccagac cactaccgaa aggcccttca tccagaagct gttcagacca
     6301 gtggctgccg acggtcagct gcacactctg ggcgacctgc tgaaggaggt ctgcccatca
     6361 gctatcgacc ctgaggacgg agaaaagaag aaccaggtca tgatccacgg aatcgagcca
     6421 atgctggaaa cccctctgca atggctgtcc gaacacctct cctacccaga caacttcctc
     6481 cacatttcca ttattcccca acctacggac taaaagcttg tcgagaagta ctagaggatc
     6541 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     6601 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     6661 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6721 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga
     6781 tcactgcttg agcctagaag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     6841 tgctgccacc gctgagcaat aactatcata accatttaaa taaactggat actattgcac
     6901 gtttaaatcg acctactccg gaatattaat agatcatgga gataattaaa atgataacca
     6961 tctcgcaaat aaataagtat tttactgttt tcgtaacagt tttgtaataa aaaaacctat
     7021 aaatattccg gattattcat accgtcccac catcgggcgc ggatccatgg ccgaagagcc
     7081 ccagagcgtt ttgcagttgc ccacaagcat tgccgcagga ggcgaaggac tcacagatgt
     7141 tagccccgag actaccactc ctgagccacc ttcttcagcc gctgtctcac ctggtactga
     7201 agagcctgct ggtgacacta agaagaagat cgacatcctg ctgaaggctg tcggtgacac
     7261 tcctatcatg aagaccaaga agtgggctgt ggagaggacc cgtactatcc agggtctgat
     7321 cgacttcatc aagaagttcc tgaagctggt ggcttctgaa cagctgttca tctacgtgaa
     7381 ccagtccttc gctccatctc ctgaccagga agtgggtact ctgtacgagt gtttcggaag
     7441 cgacggcaag ttggttctcc attactgcaa atcacaagcg tggggctaaa agcttgtcga
     7501 gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     7561 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     7621 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     7681 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     7741 tcatgtctgg atctgatcac tgcttgagcc tagaagatcc ggctgctaac aaagcccgaa
     7801 aggaagctga gttggctgct gccaccgctg agcaataact atcataacca tttaaataaa
     7861 tctatatctc aatcggggtt acgtcgatac cccaccgaga ccccattggg accaatacgc
     7921 ccgcgtttct tccttttccc caccccaacc cccaagttcg ggtgaaggcc cagggctcgc
     7981 agccaacgtc ggggcggcaa gccctgccat agccactacg ggttaattaa aaactggata
     8041 ctattgcacg tttaaacagg agatccgaac cagataagtg aaatctagtt ccaaactatt
     8101 ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca tctattttgt
     8161 cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt cccaactatt
     8221 ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa catcctgcca
     8281 actccatgtg acaaaccgtc atcttcggct acttt
//
