LOCUS       Exported                7241 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses mCherry-EGFP-LC3B wild-type in mammalian cells. 
            Fluorescent tandem reporter for autophagosomes. High expression 
            driven by CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pDEST-CMV mCherry-GFP-LC3B WT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  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 7241)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7241
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             860..1567
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1005..1023)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1273..1292
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    1595..1619
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1628..1637
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1634..2350
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1679..1700)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1940..1959)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2287..2308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2372..2396
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2399..2773
                     /codon_start=1
                     /gene="MAP1LC3B"
                     /product="human LC3 protein"
                     /label=LC3B
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD
                     EDGFLYMVYASQETFGMKLSV"
     protein_bind    complement(2778..2802)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2834..2851)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2840..3064
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3110..3538
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3197..3216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3407..3428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3547..3567)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3552..3881
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3732..3867
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3794..3813
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3948..4742
                     /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(4002..4021)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4612..4631
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4916..5037
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4953..4972)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5007..5026
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5086..5102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5086..5102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5099..5121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5110..5126
                     /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(5134..5164)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5179..5200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5317..5334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5488..6076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5568..5587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6247..7107)
                     /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     6870..6889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7108..7212)
                     /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 accgtcacaa
      841 ttgatctggt accgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
      901 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
      961 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     1021 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     1081 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     1141 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     1201 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     1261 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     1321 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     1381 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     1441 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     1501 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     1561 gtacaagtcc ggactcagat ccgctggccc aggaacaagt ttgtacaaaa aagcaggctt
     1621 tccggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1681 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1741 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1801 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1861 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1921 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1981 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2041 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2101 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2161 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2221 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2281 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactcacg gcatggacga
     2341 gctctacaaa agcggttccg gaccggatca aacaagtttg tacaaaaaag caggcttaat
     2401 gccgtcggag aagaccttca agcagcggcg caccttcgaa caaagagtag aagatgtccg
     2461 acttattcga gagcagcatc caaccaaaat cccggtgata atagaacgat acaagggtga
     2521 gaagcagctt cctgttctgg ataaaacaaa gttccttgta cctgaccatg tcaacatgag
     2581 tgagctcatc aagataatta gaaggcgctt acagctcaat gctaatcagg ccttcttcct
     2641 gttggtgaac ggacacagca tggtcagcgt ctccacacca atctcagagg tgtatgagag
     2701 tgagaaagat gaagatggat tcctgtacat ggtctatgcc tcccaggaga cgttcgggat
     2761 gaaattgtca gtgtaatacc cagctttctt gtacaaagtg gtgataatta attaagataa
     2821 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2881 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2941 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3001 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3061 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3121 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3181 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3241 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3301 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3361 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3421 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3481 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3541 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3601 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3661 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3721 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3781 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3841 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3901 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3961 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4021 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4081 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4141 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4201 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4261 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4321 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4381 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4441 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4501 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4561 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4621 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4681 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4741 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4801 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4861 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4921 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4981 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5041 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5101 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5161 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5221 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6361 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7201 aggggttccg cgcacatttc cccgaaaagt gccacctgac g
//
