LOCUS       sequence-332048-        6515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conjugatable form of LC3B lacking 5 C-terminal amino acids (MKLSV), 
            with C-terminal Glycine120 exposed for lipidation reaction..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6515)
  AUTHORS   Zaffagnini G, Savova A, Danieli A, Romanov J, Tremel S, Ebner M, 
            Peterbauer T, Sztacho M, Trapannone R, Tarafder AK, Sachse C, 
            Martens S
  TITLE     p62 filaments capture and present ubiquitinated cargos for 
            autophagy.
  JOURNAL   EMBO J. 2018 Mar 1;37(5). pii: embj.201798308. doi: 
            10.15252/embj.201798308. Epub 2018 Jan 17.
  PUBMED    29343546
REFERENCE   2  (bases 1 to 6515)
  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: "EMBO J."; 
            date: "2018-03-1"; volume: "37(5). pii: embj.201798308. doi"; pages:
            " 10.15252/embj.201798308. Epub 2018 Jan 17"
COMMENT     Sequence Label: 6xHis-TEV-mCherry-LC3B-Gly (Microtubule-associ...
FEATURES             Location/Qualifiers
     source          1..6515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     355..374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     608..625
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    640..779
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(705..724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     865..887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(881..1072)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1644..2726)
                     /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     2688..2707
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2727..2804)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3010..3029
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3117..3135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3136..3160
                     /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             3175..3197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3216..3233
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3246..3266
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3267..3971
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3409..3427)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3677..3696
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        4445..4463
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4464..4488
                     /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             4597..4641
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4679..4697)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4693..4740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4777..5232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4864..4883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5074..5095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5347..6138,6139..6207))
                     /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     5970..5989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6208..6300)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6381..6515,1..454)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
       61 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      481 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      541 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      601 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
      661 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
      721 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
      781 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      841 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      901 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
      961 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     1021 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     1081 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     1141 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     1201 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     1261 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     1321 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     1381 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     1441 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     1501 gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc
     1561 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     1621 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     1741 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     1801 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     1861 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     1921 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     1981 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     2041 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     2101 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     2161 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     2221 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     2281 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     2341 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     2401 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     2461 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     2521 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     2581 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     2641 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     2701 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     2761 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     2821 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     2881 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     2941 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     3001 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     3061 gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat
     3121 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     3181 taactttaag aaggagatat accatgggca gcagccatca ccatcatcac cacagccagg
     3241 atccggaaaa cctgtatttt cagggagtga gcaagggcga ggaggataac atggccatca
     3301 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     3361 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     3421 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     3481 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     3541 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     3601 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     3661 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     3721 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     3781 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     3841 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     3901 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     3961 agctgtacaa gtccggactc agatctcgag ctcaagcttc gaattctgca gtcgacatgc
     4021 cgtcggagaa gaccttcaag cagcgccgca ccttcgaaca aagagtagaa gatgtccgac
     4081 ttattcgaga gcagcatcca accaaaatcc cggtgataat agaacgatac aagggtgaga
     4141 agcagcttcc tgttctggat aaaacaaagt tccttgtacc tgaccatgtc aacatgagtg
     4201 agctcatcaa gataattaga aggcgcttac agctcaatgc taatcaggcc ttcttcctgt
     4261 tggtgaacgg acacagcatg gtcagcgtct ccacaccaat ctcagaggtg tatgagagtg
     4321 agaaagatga agatggattc ctgtacatgg tctatgcctc ccaggagacg ttcgggtgag
     4381 cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg
     4441 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc atcttagtat
     4501 attagttaag tataagaagg agatatacat atggcagatc tcaattggat atcggccggc
     4561 cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc tgcgaaattt
     4621 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
     4681 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     4741 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     4801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5221 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
     5281 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5341 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5401 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5461 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5521 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5581 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5641 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5701 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5761 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5821 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5881 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5941 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6001 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6061 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6121 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6181 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     6241 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6301 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6361 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6421 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6481 tttccgaagg taactggctt cagcagagcg cagat
//
