LOCUS       sequence-439216-        7075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flag tagged LC3B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7075)
  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 7075)
  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: pKH151_PB_flag-LC3B_wt_IRES-BFP
FEATURES             Location/Qualifiers
     source          1..7075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1482..1516
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    2279..2400
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2290..2309)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2344..2363
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2409..3110)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLHDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3122..3674
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3284..3303)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3513..3530
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(3693..4067)
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFAMKLSV"
     CDS             complement(4068..4133)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     LTR             4164..4432
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(4445..4656)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     repeat_region   5109..5171
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5301..5318)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5301..5317)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5310..5332)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5459..5914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5546..5565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5756..5777
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5940..6044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6045..6113,6114..6905)
                     /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(6263..6282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg cccgccgggt
     1021 aactcacggg gtatccatgt ccatttctgc ggcatccagc caggataccc gtcctcgctg
     1081 acgtaatatc ccagcgccgc accgctgtca ttaatctgca caccggcacg gcagttccgg
     1141 ctgtcgccgg tattgttcgg gttgctgatg cgcttcgggc tgaccatccg gaactgtgtc
     1201 cggaaaagcc gcgacgaact ggtatcccag gtggcctgaa cgaacagttc accgttaaag
     1261 gcgtgcatgg ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg ctcaacgtca
     1321 atgcagcagc agtcatcctc ggcaaactct ttccatgccg cttcaacctc gcgggaaaag
     1381 gcacgggctt cttcctcccc gatgcccaga tagcgccagc ttgggcgatg actgagccgg
     1441 aaaaaagacc cgacgatatg atcctgatgc agctagatta accctagaaa gatagtctgc
     1501 gtaaaattga cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg
     1561 tcgctgtgca tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca
     1621 atgcggtaag tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga
     1681 ttatctttta cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt
     1741 tctacttacg tgataactta ttatatatat attttcttgt tatagatatc agggacagcc
     1801 cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag
     1861 ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg
     1921 gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct
     1981 gctctttgag cctgcagaca cctgggggga tacggggaaa aagccatacc aatgggccct
     2041 aaaaaaggaa tccagtcaat tccggggcta aacctggctg ccactgtttc tttagggact
     2101 tcgttcctgt gaggacagga aaccctgcaa cctggttgac cagagcaagg gatataagaa
     2161 cccagtttgg gaacactgct gtttaaatat taaacaggga tgtcttgtcc cccggaaggg
     2221 agaccagatg agcttctgat agactaattg acatcatttg agtcaattcc agacatgata
     2281 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     2341 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     2401 tagttaactt aattaagctt gtgccccagt ttgctaggga ggtcgcagta tctggccact
     2461 gccacctcgt gctgctcgac gtaggtctcg ttgttggcct ccttgattct ttccagtctg
     2521 tagtccacat agtagacgcc aggcatcttg aggttcttag cgggtttctt ggatctatat
     2581 gtggtcttga tgtttgcgat cagatggctc ccgcccacga gcttcagggc catgtcgttt
     2641 ctgccttcca ggccgccgtc agcggggtac agcgtctcgg tgaaggcctc ccagccgagt
     2701 gttttcttct gcatcacagg gccgttggat gtgaagttca cccctctgat cttgacgttg
     2761 tagatgaggc agccgtcatg gaggctggtg tcctgggtag cggtcagcac gcccccgtct
     2821 tcgtatgtgg tgactctctc ccatgtgaag ccctcaggga aggactgctt gaagaagtcg
     2881 gggatgccct gggtgtggtt gatgaaggtc ttgctgccgt agaggaagct agtagccagg
     2941 atgtcgaagg cgaaggggag agggccgccc tcgaccacct tgattctcat ggtctgggtg
     3001 ccctcgtagg gcttgccttc gccctcggat gtgcacttga agtgatggtt gtccacggtg
     3061 ccctccatgt acagcttcat gtgcatgttc tccttaatca gctcgctcat ggttgtggcc
     3121 atattatcat cgtgtttttc aaaggaaaac cacgtccccg tggttcgggg ggcctagacg
     3181 tttttttaac ctcgactaaa tacatgtaaa gcatgtgcac cgaggcccca gatcagatcc
     3241 catacaatgg ggtaccttct gggcatcctt cagccccttg ttgaatacgc ttgaggagag
     3301 ccatttgact ctttccacaa ctatccaact cacaacgtgg cactggggtt gtgccgcctt
     3361 tgcaggtgta tcttatacac gtggcttttg gccgcagagg cacctgtcgc caggtggggg
     3421 gttccgctgc ctgcaaaggg tcgctacaga cgttgtttgt cttcaagaag cttccagagg
     3481 aactacttcc ttcacgacat tcaacagacc ttgcattcct ttggcgagag gggaaagacc
     3541 cctaggaatg ctcgtcaaga agacagggcc aggtttccgg gccctcacat tgccaaaaga
     3601 cggcaatatg gtggaaaata acatatagac aaacgcacac cggccttatt ccaagcggct
     3661 tcggccagta acgttagggg gggggggcgt tacactgaca atttcatcgc gaacgtctcc
     3721 tgggaggcat agaccatgta caggaatcca tcttcatctt tctcactctc atacacctct
     3781 gagattggtg tggagacgct gaccatgctg tgtccgttca ccaacaggaa gaaggcctga
     3841 ttagcattga gctgtaagcg ccttctaatt atcttgatga gctcactcat gttgacatgg
     3901 tcaggtacaa ggaactttgt tttatccaga acaggaagct gcttctcacc cttgtatcgt
     3961 tctattatca ccgggatttt ggttggatgc tgctctcgaa taagtcggac atcttctact
     4021 ctttgttcga aggtgcggcg ctgcttgaag gtcttctccg acggcatctt gtcatcgtca
     4081 tccttgtagt cgatgtcatg atccttgtaa tcaccgtcat ggtccttgta gtccattcta
     4141 gagtaggcgc cggtcacagc ttggatctgt aacggcgcag aacagaaaac gaaacaaaga
     4201 cgtagagttg agcaagcagg gtcaggcaaa gcgtggagag ccggctgagt ctaggtaggc
     4261 tccaagggag cgccggacaa aggcccggtc tcgacctgag ctttaaactt acctagacgg
     4321 cggacgcagt tcaggaggca ccacaggcgg gaggcggcag aacgcgactc aaccggcgtg
     4381 gatggcggcc tcaggtaggg cggcgggcgc gtgaaggaga gatgcgagcc cctcgaagct
     4441 tcagctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac
     4501 ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt
     4561 cccagtttac cccgcgccac cttctctagg cacccgttca attgccgacc cctcccccca
     4621 acttctcggg gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagcgatcgc
     4681 agatccttgg ccgtggaggc cttttccccg tatcccccca ggtgtctgca ggctcaaaga
     4741 gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc
     4801 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc
     4861 ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg
     4921 ggggctgtcc ctcataaaag ttttgttact ttatagaaga aattttgagt ttttgttttt
     4981 tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag tatgtaagtg
     5041 taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat atacagaccg
     5101 ataaaacaca tgcgtcaatt ttacacatga ttatctttaa cgtacgtcac aatatgatta
     5161 tctttctagg gttaatctag ctgcgtgttc tgcagcgtgt cgagcatctt catctgctcc
     5221 atcacgctgt aaaacacatt tgcaccgcga gtctgcccgt cctccacggg ttcaaaaacg
     5281 tgaatgaacg aggcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     5341 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     5401 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     5461 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5521 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5581 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5641 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5701 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5761 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5821 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5881 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5941 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6001 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6061 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6121 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6181 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6241 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6301 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6361 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6421 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6481 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6541 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6601 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6661 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6721 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6781 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6841 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6901 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6961 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7021 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
