LOCUS       sequence-252871-        7390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7390)
  AUTHORS   Tanida I, Yamaji T, Ueno T, Ishiura S, Kominami E, Hanada K
  TITLE     Consideration about negative controls for LC3 and expression vectors
            for four colored fluorescent protein-LC3 negative controls.
  JOURNAL   Autophagy. 2008 Jan 1. 4(1):131-4.
  PUBMED    18000393
REFERENCE   2  (bases 1 to 7390)
  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. 
            2008 Jan 1. 4(1):131-4."
COMMENT     Sequence Label: pEX-GFP-hLC3WT
FEATURES             Location/Qualifiers
     source          1..7390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          940..1956
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1880..1902
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1964..1983
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2044..2053
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2050..2052,2053..2055,2056..2766)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2050..2052,2053..2055,2056..2766)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2095..2116)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2356..2375)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2703..2724
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2782..3156
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFGMKLSV"
     primer_bind     complement(3259..3278)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3324..3379
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3378..3397)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3740..3756)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3740..3756)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3753..3775)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3764..3780
                     /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(3788..3794,3795..3812,3813..3818))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3833..3854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3913..4108
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3959..4094
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4114..4248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4151..4170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4205..4224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4316..4333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4487..5075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4567..4586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5246..6037,6038..6106))
                     /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     5869..5888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6107..6211)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6523..7326
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6586..6605)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7196..7215
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7333..7381
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggncgcgcc ccrggggggg gggggggggg gggggggggg
      541 gggggggggg gggggggggg gggggggggg gggggnnggg gggggggggg gggggggggg
      601 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
      661 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
      721 kkkkggngkk kkkkkgkkkk gkkkrkkkkk gggggggggg gggggggggg gggggggggg
      781 gggcgcgcgc ggggsggggs ggggcggggc gaggggcggg gcggggcgag gcggagaggt
      841 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      901 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      961 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     1021 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
     1081 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
     1141 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
     1201 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
     1261 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
     1321 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
     1381 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
     1441 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
     1501 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag
     1561 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
     1621 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
     1681 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
     1741 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     1801 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     1861 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     1921 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     1981 gtgctgtctc atcattttgg caaagaattc ttaagggtac cgatatcgct agcgctaccg
     2041 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2101 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2161 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2221 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2281 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2341 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2401 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2461 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2521 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2581 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2641 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2701 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2761 tacaagtccg gactcagatc tatgccgtcg gagaagacct tcaagcagcg ccgcaccttc
     2821 gaacaaagag tagaagatgt ccgacttatt cgagagcagc atccaaccaa aatcccggtg
     2881 ataatagaac gatacaaggg tgagaagcag cttcctgttc tggataaaac aaagttcctt
     2941 gtacctgacc atgtcaacat gagtgagctc atcaagataa ttagaaggcg cttacagctc
     3001 aatgctaatc aggccttctt cctgttggtg aacggacaca gcatggtcag cgtctccaca
     3061 ccaatctcag aggtgtatga gagtgagaaa gatgaagatg gattcctgta catggtctac
     3121 gcctcccagg agacgttcgg gatgaaattg tcagtgtaag tcgagcccgg gagctcaatt
     3181 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     3241 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     3301 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     3361 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     3421 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     3481 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     3541 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     3601 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     3661 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc
     3721 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3781 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3841 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3901 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3961 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4021 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4081 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     4141 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4201 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     4261 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4321 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4381 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4441 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4501 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4561 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4621 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4681 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4741 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4801 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4861 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4921 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4981 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5041 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5101 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5161 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5221 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5281 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5341 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5401 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5461 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5521 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5581 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5641 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5701 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5761 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5821 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5881 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5941 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6001 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6061 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6121 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6181 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt
     6241 cgagcagtgt ggttttcaag aggaagcaaa aagcctctcc acccaggcct ggaatgtttc
     6301 cacccaatgt cgagcagtgt ggttttgcaa gaggaagcaa aaagcctctc cacccaggcc
     6361 tggaatgttt ccacccaatg tcgagcaaac cccgcccagc gtcttgtcat tggcgaatta
     6421 attcgaacac gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg
     6481 tgacgcgtgt ggcctcgaac accgagcgac cctgcagcca atatgggatc ggccattgaa
     6541 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6601 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6661 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     6721 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6781 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6841 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6901 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6961 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     7021 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat
     7081 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     7141 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     7201 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     7261 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     7321 ttctgagggg atcggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     7381 cggatccgtc
//
