LOCUS       sequence-331461-        6820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-LC3-RFP-LC3-Delta-G in zebrafish to measure autophagic
            flux.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6820)
  AUTHORS   Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, 
            Kodama A, Ishihara T, Mizushima T, Mizushima N
  TITLE     An Autophagic Flux Probe that Releases an Internal Control.
  JOURNAL   Mol Cell. 2016 Nov 17;64(4):835-849. doi: 
            10.1016/j.molcel.2016.09.037. Epub 2016 Nov 3.
  PUBMED    27818143
REFERENCE   2  (bases 1 to 6820)
  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"; doi: 
            "10.1016/j.molcel.2016.09.037"; journalName: "Mol Cell"; date: 
            "2016-11-17- 17"; volume: "64"; issue: "4"; pages: "835-849"
COMMENT     Sequence Label: pT2AL200R150G-EGFP-LC3B-RFP-LC3B-Delta-G
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..39
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        21..39
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     76..92
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     76..92
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(126..137,138..325)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     intron          856..1428
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             join(1498..1500,1501..1503,1504..2214)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1498..1500,1501..1503,1504..2214)
                     /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(1543..1564)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1804..1823)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2151..2172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2638..3312
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2638..3312
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2768..2786)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3036..3055
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3230..3253
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3718..3852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3742..3761)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3796..3815
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3974..4111,4112..4123)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4153..4172)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4154..4172)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4182..4199)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4182..4198)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4510..4531
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4694..4798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4799..4867,4868..5659)
                     /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     complement(5017..5036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5830..6418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6319..6338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6572..6589
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6706..6727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6742..6747,6748..6765,6766..6772)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6780..6796
                     /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)."
     primer_bind     6785..6807
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6804..6820
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6804..6820
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac
       61 cgcggtggcg gccgctctag aactagtgga tctgctgggc ttgctgaagg tagggggtca
      121 agaaccagag gtgtaaagta cttgagtaat tttacttgat tactgtactt aagtattatt
      181 tttggggatt tttactttac ttgagtacaa ttaaaaatca atacttttac ttttacttaa
      241 ttacattttt ttagaaaaaa aagtactttt tactccttac aattttattt acagtcaaaa
      301 agtacttatt ttttggagat cacttgggcc cggctcgagc agggggatca tctaatcaag
      361 cacaaataag gggcgtgtaa cacaaaagcc agcgaccctt tccaatgcaa atcaaacttg
      421 caattctttg ccgtttttat catttaagtg tcggcttaag gtccactatc agatgtaaac
      481 agccttatct aacaaaggta tcattacatt ctgaaattct caggcatgca agctagctta
      541 tgacgcacta gggagtgcca cccttccttt cgccctaact tcgtgataac tcgcgcgttt
      601 cactcaacag ctgcatccgc cctagtgcta ctgggagttg tagtatacaa gacgcttaca
      661 ggctgaatgt tctgtcaaga ccccgcctct agcactttgg gaattctgga cttgatgatg
      721 tcatggttaa tccccgccca gtagaggcgg ctatataaag ggtggttaag gcccggttcg
      781 ctctcttcct caccgggtct gcggcgagtt ctagctgaag cttcctgcag gtcgaccgat
      841 cctgagaact tcagggtgag tttggggacc cttgattgtt ctttcttttt cgctattgta
      901 aaattcatgt tatatggagg gggcaaagtt ttcagggtgt tgtttagaat gggaagatgt
      961 cccttgtatc accatggacc ctcatgataa ttttgtttct ttcactttct actctgttga
     1021 caaccattgt ctcctcttat tttcttttca ttttctgtaa ctttttcgtt aaactttagc
     1081 ttgcatttgt aacgaatttt taaattcact tttgtttatt tgtcagattg taagtacttt
     1141 ctctaatcac ttttttttca aggcaatcag ggtatattat attgtacttc agcacagttt
     1201 tagagaacaa ttgttataat taaatgataa ggtagaatat ttctgcatat aaattctggc
     1261 tggcgtggaa atattcttat tggtagaaac aactacaccc tggtcatcat cctgcctttc
     1321 tctttatggt tacaatgata tacactgttt gagatgagga taaaatactc tgagtccaaa
     1381 ccgggcccct ctgctaacca tgttcatgcc ttcttctctt tcctacagct cctgggcaac
     1441 gtgctggttg ttgtgctgtc tcatcatttt ggcaaagaat tcctcgacgg atccaccatg
     1501 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1561 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1621 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1681 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1741 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1801 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1861 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1921 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1981 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2041 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2101 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2161 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtggc
     2221 ggtggctcgg gcggtggtgg gtcgggtggc ggcggatcta tcgatgaatt gaattccatg
     2281 ccgtccgaga agaccttcaa acagcgccgg agcttcgaac aaagagtgga agatgtccgg
     2341 ctcatccggg agcagcaccc caccaagatc ccagtgatta tagagcgata caagggtgag
     2401 aagcagctgc ccgtcctgga caagaccaag ttccttgtac ctgatcacgt gaatatgagc
     2461 gaactcatca agataattag aaggcgcctg cagctcaatg ctaaccaagc cttcttcctc
     2521 ctggtgaatg ggcacagcat ggtgagtgtg tccacaccca tctctgaagt gtacgagagc
     2581 gagagagatg aagacggctt cctgtacatg gtctatgcct cccaggagac gttcgggatg
     2641 gcctcctccg aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc
     2701 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     2761 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
     2821 tcccctcagt tccagtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     2881 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     2941 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     3001 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     3061 ggctgggagg cctccaccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     3121 aagatgaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac
     3181 atggccaaga agcccgtgca gctgcccggc gcctacaaga ccgacatcaa gctggacatc
     3241 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac
     3301 tccaccggcg ccatcgatga attgaattcc atgccgtccg agaagacctt caaacagcgc
     3361 cggagcttcg aacaaagagt ggaagatgtc cggctcatcc gggagcagca ccccaccaag
     3421 atcccagtga ttatagagcg atacaagggt gagaagcagc tgcccgtcct ggacaagacc
     3481 aagttccttg tacctgatca cgtgaatatg agcgaactca tcaagataat tagaaggcgc
     3541 ctgcagctca atgctaacca agccttcttc ctcctggtga atgggcacag catggtgagt
     3601 gtgtccacac ccatctctga agtgtacgag agcgagagag atgaagacgg cttcctgtac
     3661 atggtctatg cctcccagga gacgttctaa ggatcccccg ggtcgcgaat cgatgatgat
     3721 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     3781 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     3841 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     3901 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct
     3961 ctagatcaga tctaatactc aagtacaatt ttaatggagt acttttttac ttttactcaa
     4021 gtaagattct agccagatac ttttactttt aattgagtaa aattttccct aagtacttgt
     4081 actttcactt gagtaaaatt tttgagtact ttttacacct ctgtcaagaa ccatatgccg
     4141 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     4201 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4261 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4321 agcctgaatg gcgaatggga cgcgccctcc gtcaagctct aaatcggggg ctccctttag
     4381 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4441 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4501 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4561 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4621 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4741 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4801 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4861 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4921 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4981 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5041 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5101 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5161 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5221 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5281 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5341 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5401 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5461 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5521 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5581 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5641 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5701 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5941 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6481 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6541 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6601 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     6661 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6721 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6781 tgtgagcgga taacaatttc acacaggaaa cagctatgac
//
