LOCUS       sequence-431710-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flag tagged LDS mutant LC3B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  AUTHORS   Hickey KL, Swarup S, Smith IR, Paoli JC, Miguel Whelan E, Paulo JA, 
            Harper JW
  TITLE     Proteome census upon nutrient stress reveals Golgiphagy membrane 
            receptors.
  JOURNAL   Nature. 2023 Nov;623(7985):167-174. doi: 10.1038/s41586-023-06657-6.
            Epub 2023 Sep 27.
  PUBMED    37757899
REFERENCE   2  (bases 1 to 7074)
  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"; doi: 
            "10.1038/s41586-023-06657-6"; journalName: "Nature"; date: 
            "2023-11"; volume: "623"; issue: "7985"; pages: "167-174"
COMMENT     Sequence Label: pKH152_PB_flag-LC3B_LDSmut_IRES-BFP
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(310..1101,1102..1170))
                     /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     933..952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1171..1275)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1301..1756)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1438..1459)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1650..1669
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1883..1905
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1897..1914
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1898..1914
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2044..2106
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2559..2770
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2783..3051
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3082..3147
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3148..3522
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTAFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFAMKLSV"
     misc_feature    3540..4092
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3684..3701)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3911..3930
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4104..4805
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4814..4935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4851..4870)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4905..4924
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5698..5732
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6223..6239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6223..6239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6236..6258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6247..6263
                     /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)."
     promoter        complement(join(6271..6277,6278..6295,6296..6301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6316..6337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6454..6471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6625..7074,1..139))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6705..6724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
       61 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      121 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      181 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      241 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      301 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      361 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      421 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      481 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      541 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      601 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      661 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      721 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      781 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      841 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      901 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      961 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1021 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1081 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1141 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1261 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     1321 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     1381 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     1441 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     1501 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     1561 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     1621 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     1681 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     1741 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     1801 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     1861 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     1921 gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg
     1981 ttttacagcg tgatggagca gatgaagatg ctcgacacgc tgcagaacac gcagctagat
     2041 taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgt gtaaaattga
     2101 cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag
     2161 ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt
     2221 atttatttat taaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta
     2281 tgagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
     2341 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     2401 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     2461 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc
     2521 acggccaagg atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2581 agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg
     2641 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2701 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2761 cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac
     2821 ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc
     2881 ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc
     2941 ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct
     3001 tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg
     3061 accggcgcct actctagaat ggactacaag gaccatgacg gtgattacaa ggatcatgac
     3121 atcgactaca aggatgacga tgacaagatg ccgtcggaga agaccttcaa gcagcgccgc
     3181 accttcgaac aaagagtaga agatgtccga cttattcgag agcagcatcc aaccaaaatc
     3241 ccggtgataa tagaacgata caagggtgag aagcagcttc ctgttctgga taaaacagcg
     3301 ttccttgtac ctgaccatgt caacatgagt gagctcatca agataattag aaggcgctta
     3361 cagctcaatg ctaatcaggc cttcttcctg ttggtgaacg gacacagcat ggtcagcgtc
     3421 tccacaccaa tctcagaggt gtatgagagt gagaaagatg aagatggatt cctgtacatg
     3481 gtctatgcct cccaggagac gttcgcgatg aaattgtcag tgtaacgccc ccccccctaa
     3541 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     3601 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     3661 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     3721 gaaggaagta gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     3781 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     3841 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     3901 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     3961 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtatttagtc
     4021 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     4081 acgatgataa tatggccaca accatgagcg agctgattaa ggagaacatg cacatgaagc
     4141 tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca
     4201 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     4261 tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca
     4321 cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag
     4381 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     4441 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga
     4501 tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg
     4561 gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa
     4621 acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct
     4681 actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc
     4741 agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta
     4801 attaagttaa ctaaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4861 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4921 catcaatgta tcttatcatg tctggaattg actcaaatga tgtcaattag tctatcagaa
     4981 gctcatctgg tctcccttcc gggggacaag acatccctgt ttaatattta aacagcagtg
     5041 ttcccaaact gggttcttat atcccttgct ctggtcaacc aggttgcagg gtttcctgtc
     5101 ctcacaggaa cgaagtccct aaagaaacag tggcagccag gtttagcccc ggaattgact
     5161 ggattccttt tttagggccc attggtatgg ctttttcccc gtatcccccc aggtgtctgc
     5221 aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac cttccccgtg
     5281 cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg gaccggagcg
     5341 gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta catccctggg
     5401 ggctttgggg gggggctgtc cctgatatct ataacaagaa aatatatata taataagtta
     5461 tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc
     5521 acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg
     5581 acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct
     5641 aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat
     5701 gcgtcaattt tacgcagact atctttctag ggttaatcta gctgcatcag gatcatatcg
     5761 tcgggtcttt tttccggctc agtcatcgcc caagctggcg ctatctgggc atcggggagg
     5821 aagaagcccg tgccttttcc cgcgaggttg aagcggcatg gaaagagttt gccgaggatg
     5881 actgctgctg cattgacgtt gagcgaaaac gcacgtttac catgatgatt cgggaaggtg
     5941 tggccatgca cgcctttaac ggtgaactgt tcgttcaggc cacctgggat accagttcgt
     6001 cgcggctttt ccggacacag ttccggatgg tcagcccgaa gcgcatcagc aacccgaaca
     6061 ataccggcga cagccggaac tgccgtgccg gtgtgcagat taatgacagc ggtgcggcgc
     6121 tgggatatta cgtcagcgag gacgggtatc ctggctggat gccgcagaaa tggacatgga
     6181 taccccgtga gttacccggc gggcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     6241 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6301 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6361 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6421 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6481 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6541 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6601 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6661 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6721 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6781 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6841 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6901 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6961 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7021 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatt
//
