LOCUS       sequence-373594-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression and purification of 
            6xHis-TEV-LC3BGlydelta5C. Internal reference: SMC893..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  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 5777)
  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: pET-Duet1-6xHis-TEV-LC3BGlydelta5C
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        574..592
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    593..617
                     /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)."
     CDS             726..770
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(808..826)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      822..869
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      906..1361
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(993..1012)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1203..1224
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1476..2267,2268..2336))
                     /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     2099..2118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2337..2429)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2510..3098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2999..3018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3252..3269
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3284..3423
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3349..3368)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3509..3531
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3525..3716)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4288..5370)
                     /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     5332..5351
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5371..5448)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5654..5673
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5761..5777,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cggaaaacct
      121 gtattttcag ggaatgccgt cggagaagac cttcaagcag cgccgcacct tcgaacaaag
      181 agtagaagat gtccgactta ttcgagagca gcatccaacc aaaatcccgg tgataataga
      241 acgatacaag ggtgagaagc agcttcctgt tctggataaa acaaagttcc ttgtacctga
      301 ccatgtcaac atgagtgagc tcatcaagat aattagaagg cgcttacagc tcaatgctaa
      361 tcaggccttc ttcctgttgg tgaacggaca cagcatggtc agcgtctcca caccaatctc
      421 agaggtgtat gagagtgaga aagatgaaga tggattcctg tacatggtct atgcctccca
      481 ggagacgttc gggtgagtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      541 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      601 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      661 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
      721 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
      781 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      841 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
      901 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      961 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1021 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1081 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1141 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1201 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1261 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1321 atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg
     1381 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1441 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1501 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1561 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1621 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1681 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1741 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1801 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1861 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1921 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1981 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2041 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2101 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2161 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2221 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2281 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2341 ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2401 atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt
     2461 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2521 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2581 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2641 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2701 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2761 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2821 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2881 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2941 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3001 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3061 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3121 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3181 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3241 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3301 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3361 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3421 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3481 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3541 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3601 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3661 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3721 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3781 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3841 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3901 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3961 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4021 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4081 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4141 agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag
     4201 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4261 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4321 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4381 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4441 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4501 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4561 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4621 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4681 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4741 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4801 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4861 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4921 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4981 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5041 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5101 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5161 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5221 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5281 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5341 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5401 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5461 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5521 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5581 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5641 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5701 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
     5761 taatacgact cactata
//
