LOCUS       sequence-243913-        5106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-LC3A wild-type in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5106)
  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 5106)
  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: pCMV 3xFLAG-LC3A WT
FEATURES             Location/Qualifiers
     source          1..5106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(22..610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(102..121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(781..1572,1573..1641))
                     /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     1404..1423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1642..1746)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1773..2228
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1860..1879)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2070..2091
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2354..2376
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2368..2385
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2369..2385
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2546..2925
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2926..3129
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3080..3100
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3166..3231
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3235..3259
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3262..3624
                     /codon_start=1
                     /gene="MAP1LC3A"
                     /product="human LC3 protein"
                     /label=LC3A
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSDRPFKQRRSFADRCKEVQQIRDQHPSKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELVKIIRRRLQLNPTQAFFLLVNQHSMVSVSTPIADIYEQEKD
                     EDGFLYMVYASQETFGF"
     RBS             3312..3320
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    complement(3629..3653)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3671..4293
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3870..3889)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4317..4337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4322..4651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4502..4637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4693..4712)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4694..4712)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4726..4742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4726..4742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4739..4761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4750..4766
                     /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(4774..4780,4781..4798,4799..4804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4957..4974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
       61 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      121 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      181 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
      241 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
      301 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
      361 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
      421 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
      481 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
      541 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
      601 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
      661 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
      721 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      781 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
      841 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      901 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      961 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1021 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1081 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1141 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     1201 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1261 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1321 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1381 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1441 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     1501 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1561 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1621 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1681 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1741 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc
     1801 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1861 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1921 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1981 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2041 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2101 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2161 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2221 tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2281 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2341 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctgatctat
     2401 acattgaatc aatattggca attagccata ttagtcattg gttatatagc ataaatcaat
     2461 attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc
     2521 tcatgtccaa tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca
     2581 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     2641 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2701 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2761 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac
     2821 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt
     2881 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2941 cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     3001 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     3061 aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3121 agcagagctc gtttagtgaa ccgtcagaat tgatctggta ccatggacta caaagaccat
     3181 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa gcttacaagt
     3241 ttgtacaaaa aagcaggctt aatgccctca gaccggcctt tcaagcagcg gcggagcttc
     3301 gccgaccgct gtaaggaggt acagcagatc cgcgaccagc accccagcaa aatcccggtg
     3361 atcatcgagc gctacaaggg tgagaagcag ctgcccgtcc tggacaagac caagtttttg
     3421 gtcccggacc atgtcaacat gagcgagttg gtcaagatca tccggcgccg cctgcagctg
     3481 aaccccacgc aggccttctt cctgctggtg aaccagcaca gcatggtgag tgtgtccacg
     3541 cccatcgcgg acatctacga gcaggagaaa gacgaggacg gcttcctcta tatggtctac
     3601 gcctcccagg aaaccttcgg cttctaatac ccagctttct tgtacaaagt ggtgtgatct
     3661 agaggatccc gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     3721 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     3781 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     3841 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     3901 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     3961 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     4021 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     4081 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     4141 tgtccttctg attttaaaat aactatacca gcaggaggac gtccagacac agcataggct
     4201 acctggccat gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg
     4261 cgttgggtcc actcagtaga tgcctgttga attgggtacg cggccagctt ggctgtggaa
     4321 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4381 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4441 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4501 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4561 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4621 aggctttttt ggaggcctag gcttttgcaa aaagctcctc gatcgaggaa ctgaaaaacc
     4681 agaaagttaa ttccctatag tgagtcgtat taaattcgta atcatgtcat agctgtttcc
     4741 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4801 taaagcctgg ggtgcctaat gagcgagcta actcacatta attgcgttgc gctcactgcc
     4861 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4921 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5101 ccgtaa
//
