LOCUS       sequence-244026-        6467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-LC3B G120A in mammalian cells. Negative control 
            fluorescent reporter, unable to localize to autophagosomes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6467)
  AUTHORS   Agrotis A, Pengo N, Burden JJ, Ketteler R
  TITLE     Redundancy of human ATG4 protease isoforms in autophagy and 
            LC3/GABARAP processing revealed in cells.
  JOURNAL   Autophagy. 2019 Jan 21. doi: 10.1080/15548627.2019.1569925.
  PUBMED    30661429
REFERENCE   2  (bases 1 to 6467)
  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: "Autophagy. 
            2019 Jan 21. doi: 10.1080/15548627.2019.1569925."
COMMENT     Sequence Label: pDEST-CMV GFP-LC3B G120A
FEATURES             Location/Qualifiers
     source          1..6467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      854..863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             860..1576
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(860..862,863..865,866..1576)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(905..926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1166..1185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1513..1534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1598..1622
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1625..1999
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFAMKLSV"
     protein_bind    complement(2004..2028)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2060..2077)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2066..2290
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2336..2764
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2423..2442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2633..2654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2773..2793)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2778..3107
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2958..3093
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3020..3039
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3174..3968
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3228..3247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3838..3857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4142..4263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4179..4198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4233..4252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4312..4328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4312..4328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4325..4347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4336..4352
                     /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(4360..4366,4367..4384,4385..4390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4405..4426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4543..4560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4714..5302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4794..4813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5473..6264,6265..6333))
                     /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     6096..6115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6334..6438)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagaa
      841 ttgatctggt accgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      901 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      961 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1021 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1081 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1141 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1201 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1261 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1321 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1381 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1441 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1501 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat
     1561 ggacgagctc tacaaaagcg gttccggacc ggatcaaaca agtttgtaca aaaaagcagg
     1621 cttaatgccg tcggagaaga ccttcaagca gcggcgcacc ttcgaacaaa gagtagaaga
     1681 tgtccgactt attcgagagc agcatccaac caaaatcccg gtgataatag aacgatacaa
     1741 gggtgagaag cagcttcctg ttctggataa aacaaagttc cttgtacctg accatgtcaa
     1801 catgagtgag ctcatcaaga taattagaag gcgcttacag ctcaatgcta atcaggcctt
     1861 cttcctgttg gtgaacggac acagcatggt cagcgtctcc acaccaatct cagaggtgta
     1921 tgagagtgag aaagatgaag atggattcct gtacatggtc tatgcctccc aggagacgtt
     1981 cgcgatgaaa ttgtcagtgt aatacccagc tttcttgtac aaagtggtga taattaatta
     2041 agataaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2101 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2161 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2221 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2281 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2341 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2401 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2461 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2521 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2581 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2641 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2701 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2761 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2821 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2881 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2941 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3001 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3061 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3121 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3181 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3241 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3301 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3361 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3421 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3481 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3541 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3601 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3661 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3721 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3781 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3841 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3901 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3961 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4021 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4081 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4141 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4201 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4261 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4321 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4381 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4441 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4501 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4561 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4621 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4681 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4741 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4801 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4861 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4921 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4981 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5041 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5101 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5161 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5221 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5281 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5341 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5401 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5461 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5521 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5581 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5641 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5701 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5761 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5821 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5881 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5941 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6001 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6061 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6121 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6181 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6241 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6301 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6361 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6421 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacg
//
