LOCUS       sequence-244029-        6452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-LC3B G120 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6452)
  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 6452)
  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 G120
FEATURES             Location/Qualifiers
     source          1..6452
                     /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"
     protein_bind    complement(1989..2013)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2045..2062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2051..2275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2321..2749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2408..2427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2618..2639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2758..2778)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2763..3092
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2943..3078
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3005..3024
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3159..3953
                     /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(3213..3232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3823..3842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4127..4248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4164..4183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4218..4237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4297..4313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4297..4313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4310..4332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4321..4337
                     /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(4345..4351,4352..4369,4370..4375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4390..4411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4528..4545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4699..5287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4779..4798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5458..6249,6250..6318))
                     /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     6081..6100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6319..6423)
                     /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 cgggtaatac ccagctttct tgtacaaagt ggtgataatt aattaagata aacccgctga
     2041 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2101 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2161 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2221 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2281 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2341 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2401 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2461 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2521 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2581 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2641 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2701 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2761 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2821 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2881 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2941 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3001 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3061 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3121 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3181 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3241 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3301 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3361 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3421 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3481 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3541 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3601 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3661 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3721 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3781 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3841 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3901 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3961 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4021 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4081 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4141 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4201 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4261 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4321 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4381 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4441 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4501 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4561 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4621 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4681 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4741 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4801 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4861 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4921 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4981 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5041 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5101 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5161 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5221 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5281 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5341 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5401 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5461 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5521 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5581 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5641 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5701 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5761 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5821 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5881 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5941 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6001 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6061 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6121 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6181 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6241 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6301 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6361 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6421 gcgcacattt ccccgaaaag tgccacctga cg
//
