LOCUS       sequence-215720-        6339 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For  expression of the mammalian Atg8 protein LC3A without a tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6339)
  AUTHORS   Nguyen TN, Padman BS, Usher J, Oorschot V, Ramm G, Lazarou M
  TITLE     Atg8 family LC3/GABARAP proteins are crucial for 
            autophagosome-lysosome fusion but not autophagosome formation during
            PINK1/Parkin mitophagy and starvation.
  JOURNAL   J Cell Biol. 2016 Dec 19;215(6):857-874. Epub 2016 Nov 18.
  PUBMED    27864321
REFERENCE   2  (bases 1 to 6339)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2016-12-19"; volume: "215(6)"; pages: "857-874. Epub 
            2016 Nov 18"
COMMENT     Sequence Label: pMX-IY LC3A
FEATURES             Location/Qualifiers
     source          1..6339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             62..655
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    718..1075
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1134..1550
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1480..1502
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1518..1534
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1560..1934
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1872..1888
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             1988..2350
                     /codon_start=1
                     /gene="MAP1LC3A"
                     /product="human LC3 protein"
                     /label=LC3A
                     /note="Atg8 family member found on autophagosomes"
                     /translation="MPSDRPFKQRRSFADRCKEVQQIRDQHPSKIPVIIERYKGEKQLP
                     VLDKTKFLVPDHVNMSELVKIIRRRLQLNPTQAFFLLVNQHSMVSVSTPIADIYEQEKD
                     EDGFLYMVYASQETFGF"
     RBS             2038..2046
                     /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)"
     misc_feature    join(2407..2981,2982..2984,2985..2993)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2574..2591)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2801..2820
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2994..2996,2997..2999,3000..3713)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3039..3060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3300..3319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3647..3668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3884..4353
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4483..4500)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4654..5242)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4734..4753)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5406..6197,6198..6266))
                     /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     6029..6048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6267..6339,1..32))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgca
       61 taatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
      121 catggaaaaa tacataactg agaatagaaa agttcagatc aaggtcagga acagatggaa
      181 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
      241 caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
      301 gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc
      361 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
      421 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
      481 aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgattgac tgagtcgccc
      541 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
      601 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatttgggg
      661 gctcgtccgg gatcgggaga cccctgccca gggaccaccg acccaccacc gggaggtaag
      721 ctggccagca acttatctgt gtctgtccga ttgtctagtg tctatgactg attttatgcg
      781 cctgcgtcgg tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga
      841 cgagttcgga acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt
      901 ttgtggcccg acctgagtcc aaaaatcccg atcgttttgg actctttggt gcacccccct
      961 aataggaggg atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt
     1021 ctgaattttt gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca
     1081 tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga
     1141 ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc
     1201 acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc
     1261 caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg
     1321 ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg
     1381 tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta
     1441 agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga
     1501 ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc cccatatggc
     1561 catatgagat cttatatggg gcacccccgc cccttgtaaa cttccctgac cctgacatga
     1621 caagagttac taacagcccc tctctccaag ctcacttaca ggctctctac ttagtccagc
     1681 acgaagtctg gagacctctg gcggcagcct accaagaaca actggaccga ccggtggtac
     1741 ctcaccctta ccgagtcggc gacacagtgt gggtccgccg acaccagact aagaacctag
     1801 aacctcgctg gaaaggacct tacacagtcc tgctgaccac ccccaccgcc ctcaaagtag
     1861 acggcatcgc agcttggata cacgccgccc acgtgaaggc tgccgacccc gggggtggac
     1921 catcctctag actgccggat ctagctagtt aattaaggat cccagtgtgg tggtacggaa
     1981 ttccaccatg ccctcagacc ggcctttcaa gcagcggcgg agcttcgccg accgctgtaa
     2041 ggaggtacag cagatccgcg accagcaccc cagcaaaatc ccggtgatca tcgagcgcta
     2101 caagggtgag aagcagctgc ccgtcctgga caagaccaag tttttggtcc cggaccatgt
     2161 caacatgagc gagttggtca agatcatccg gcgccgcctg cagctgaacc ccacgcaggc
     2221 cttcttcctg ctggtgaacc agcacagcat ggtgagtgtg tccacgccca tcgcggacat
     2281 ctacgagcag gagaaagacg aggacggctt cctctatatg gtctacgcct cccaggaaac
     2341 cttcggcttc tgactcgagg gccggcgcgg cgcgccgcgg ccgctacgta aattccgccc
     2401 cccccccccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg
     2461 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag
     2521 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc
     2581 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg
     2641 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag
     2701 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca
     2761 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt
     2821 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc
     2881 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa
     2941 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggtga
     3001 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     3061 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     3121 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     3181 ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     3241 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     3301 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     3361 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     3421 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     3481 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     3541 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     3601 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3661 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taagtcgacg
     3721 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     3781 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg
     3841 agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac
     3901 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct
     3961 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     4021 acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt
     4081 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
     4141 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
     4201 ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata
     4261 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg
     4321 agtgattgac tacccgtcag cgggggtctt tcacatgcag catgtatcaa aattaatttg
     4381 gttttttttc ttaagtattt acattaaatg gccatagttg cattaatgaa tcggccaacg
     4441 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4501 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4561 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4621 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4681 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4741 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4801 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4861 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4921 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4981 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5041 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5101 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5161 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5221 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5281 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5341 ctagatcctt ttgcggccgg ccgcaaatca atctaaagta tatatgagta aacttggtct
     5401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6301 cagggttatt gtctcatgag cggatacata tttgaatgt
//
