LOCUS       sequence-244373-        6589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-LC3C-EGFP in mammalian cells, for monitoring ATG4 
            activity towards LC3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6589)
  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 6589)
  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 3xFLAG-LC3C-GFP
FEATURES             Location/Qualifiers
     source          1..6589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             859..924
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    928..952
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1397..1421)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1438..1440,1441..1443,1444..2157)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1438..1440,1441..1443,1444..2154)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1483..1504)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1744..1763)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2091..2112
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2184..2201)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2190..2414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2460..2888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2547..2566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2757..2778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2897..2917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2902..3231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3082..3217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3144..3163
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3298..4092
                     /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(3352..3371)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3962..3981
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4266..4387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4303..4322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4357..4376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4436..4452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4436..4452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4449..4471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4460..4476
                     /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(4484..4490,4491..4508,4509..4514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4529..4550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4667..4684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4838..5426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4918..4937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5597..6388,6389..6457))
                     /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     6220..6239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6458..6562)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag
      841 aattgatctg gtaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc
      901 gattacaagg atgacgatga caagcttaca agtttgtaca aaaaagcagg cttaatgccg
      961 cctccacaga aaatcccaag cgtcagaccc ttcaagcaga ggaaaagctt ggcaatcaga
     1021 caagaggaag ttgctggaat ccgggcaaag ttccccaaca aaatcccggt ggtagtggag
     1081 cgctacccca gggagacgtt cctgcccccg ctggacaaaa ccaagttcct ggtcccgcag
     1141 gagctgacca tgacccagtt cctcagcatc atccggagcc gcatggtcct gagagccacg
     1201 gaagcctttt acttgctggt gaacaacaag agcctggtca gcatgagcgc aaccatggca
     1261 gagatctaca gagactacaa ggatgaggat ggcttcgtgt acatgaccta cgcctcccag
     1321 gagacatttg gctgcctgga gtcagcagcc cccagggatg ggagcagcct tgaggacaga
     1381 ccctgcaatc ctctctaccc agctttcttg tacaaagtgg ttcgatctag actcgagatg
     1441 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1501 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1561 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1621 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1681 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1741 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1801 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1861 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1921 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1981 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2041 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2101 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatta
     2161 attaagataa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2221 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2281 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2341 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2401 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2461 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2521 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2581 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2641 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2701 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2761 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2821 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2881 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2941 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3001 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3061 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3121 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3181 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3241 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3301 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3361 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3421 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3481 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3541 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3601 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3661 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3721 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3781 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3841 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3901 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3961 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4021 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4081 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4141 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4201 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4261 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4321 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4381 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4441 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4501 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4621 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4681 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4741 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4801 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4861 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4921 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4981 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5041 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5101 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5161 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5221 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5281 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5341 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5401 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5461 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5521 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5581 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5641 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5701 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5761 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5821 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5881 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5941 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6001 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6061 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6121 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6181 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6241 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6301 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6361 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6421 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6481 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6541 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctga
//
