LOCUS       sequence-252420-        7165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a active-site mutant human Atg7/Apg7 C572S in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7165)
  AUTHORS   Tanida I, Tanida-Miyake E, Ueno T, Kominami E
  TITLE     The human homolog of Saccharomyces cerevisiae Apg7p is a 
            Protein-activating enzyme for multiple substrates including human 
            Apg12p, GATE-16, GABARAP, and MAP-LC3.
  JOURNAL   J Biol Chem. 2001 Jan 19;276(3):1701-6. Epub 2000 Nov 28.
  PUBMED    11096062
REFERENCE   2  (bases 1 to 7165)
  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: "J Biol 
            Chem."; date: "2001-01-19"; volume: "276(3)"; pages: "1701-6. Epub 
            2000 Nov 28"
COMMENT     Sequence Label: pCMV-hATG7CS
FEATURES             Location/Qualifiers
     source          1..7165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2741..2750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2747..2749,2750..2752,2753..3466)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2747..2749,2750..2752,2753..3463)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2792..2813)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3053..3072)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3400..3421
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3589..3710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3626..3645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3680..3699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3717..4172)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3854..3875)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4066..4085
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4199..4303
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4305..4662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4305..4496
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4328..4348)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4513..4648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4697..5491
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4697..5491
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4751..4770)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5361..5380
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5679..5698)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5723..5770
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6099..6687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6588..6607
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6862..7165
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtatcgata agcttggcac
      301 gaggcggaag ttgagcggcg gcaagaaata atggcggcag ctacggggga tcctggactc
      361 tctaaactgc agtttgcccc ttttagtagt gccttggatg ttgggttttg gcatgagttg
      421 acccagaaga agctgaacga gtatcggctg gatgaagctc ccaaggacat taagggttat
      481 tactacaatg gtgactctgc tgggctgcca gctcgcttaa cattggagtt cagtgctttt
      541 gacatgagtg ctcccacccc agcccgttgc tgcccagcta ttggaacact gtataacacc
      601 aacacactcg agtctttcaa gactgcagat aagaagctcc ttttggaaca agcagcaaat
      661 gagatatggg aatccataaa atcaggcact gctcttgaaa accctgtact cctcaacaag
      721 ttcctcctct tgacatttgc agatctaaag aagtaccact tctactattg gttttgctat
      781 cctgccctct gtcttccaga gagtttacct ctcattcagg ggccagtggg tttggatcaa
      841 aggttttcac taaaacagat tgaagcacta gagtgtgcat atgataatct ttgtcaaaca
      901 gaaggagtca cagctcttcc ttacttctta atcaagtatg atgagaacat ggtgctggtt
      961 tccttgctta aacactacag tgatttcttc caaggtcaaa ggacgaagat aacaattggt
     1021 gtatatgatc cctgtaactt agcccagtac cctggatggc ctttgaggaa ttttttggtc
     1081 ctagcagccc acagatggag tagcagtttc cagtctgttg aagttgtttg cttccgtgac
     1141 cgtaccatgc agggggcgag agacgttgcc cacagcatca tcttcgaagt gaagcttcca
     1201 gaaatggcat ttagcccaga ttgtcctaaa gcagttggat gggaaaagaa ccagaaagga
     1261 ggcatgggac caaggatggt gaacctcagt gaatgtatgg accctaaaag gttagctgag
     1321 tcatcagtgg atctaaatct caaactgatg tgttggagat tggttcctac tttagacttg
     1381 gacaaggttg tgtctgtcaa atgtctgctg cttggagccg gcaccttggg ttgcaatgta
     1441 gctaggacgt tgatgggttg gggcgtgaga cacatcacat ttgtggacaa tgccaagatc
     1501 tcctactcca atcctgtgag gcagcctctc tatgagtttg aagattgcct agggggtggt
     1561 aagcccaagg ctctggcagc agcggaccgg ctccagaaaa tattccccgg tgtgaatgcc
     1621 agaggattca acatgagcat acctatgcct gggcatccag tgaacttctc cagtgtcact
     1681 ctggagcaag cccgcagaga tgtggagcaa ctggagcagc tcatcgaaag ccatgatgtc
     1741 gtcttcctat tgatggacac cagggagagc cggtggcttc ctgccgtcat tgctgcaagc
     1801 aagagaaagc tggtcatcaa tgctgctttg ggatttgaca catttgttgt catgagacat
     1861 ggtctgaaga aaccaaagca gcaaggagct ggggacttgt gtccaaacca ccctgtggca
     1921 tctgctgacc tcctgggctc atcgcttttt gccaacatcc ctggttacaa gcttggctgc
     1981 tacttctgca atgatgtggt ggccccagga gattcaacca gagaccggac cttggaccag
     2041 cagagcactg tgagtcgtcc aggactggcc gtgattgcag gagccctggc cgtggaattg
     2101 atggtatctg ttttgcagca tccagaaggg ggctatgcca ttgccagcag cagtgacgat
     2161 cggatgaatg agcctccaac ctctcttggg cttgtgcctc accagatccg gggatttctt
     2221 tcacggtttg ataatgtcct tcccgtcagc ctggcatttg acaaatgtac agcttgttct
     2281 tccaaagttc ttgatcaata tgaacgagaa ggatttaact tcctagccaa ggtgtttaat
     2341 tcttcacatt ccttcttaga agacttgact ggtcttacat tgctgcatca agaaacccaa
     2401 gctgctgaga tctgggacat gagcgacgat gagaccatct gagatggccc cgctgtgggg
     2461 ctgacttctc cctggccgcc tgctgaggag ctctccatcg ccagagcagg actgctgacc
     2521 ccaggcctgg tgattctggg cccctcctcc ataccccgag gtctgggatt cccccctctg
     2581 ctgcccagga gtggccagtg ttcggcgttg ctcgggattc aagataccac cagttcagag
     2641 ctaaataata accttggcct tggccttgct attgacctgg gaaaaaaaaa aaaaaaaaaa
     2701 attcctgcgg ccgccaccgc gggcccggga tccaccggtc gccaccatgg tgagcaaggg
     2761 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2821 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2881 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2941 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3001 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3061 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3121 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3181 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     3241 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3301 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3361 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3421 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc
     3481 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3541 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3601 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3661 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     3721 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3781 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3841 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3901 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3961 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4021 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4081 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4141 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     4201 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4261 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     4321 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4381 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4441 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4501 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4561 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4621 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4681 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4741 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4801 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4861 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4921 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4981 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     5041 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     5101 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     5161 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     5221 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5281 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5341 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5401 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5461 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5521 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5581 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5641 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5701 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5761 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5821 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5881 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5941 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6001 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6061 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6121 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6181 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6241 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6301 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6361 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6421 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6481 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6541 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6601 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6661 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6721 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6781 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
     6841 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     6901 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     6961 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     7021 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     7081 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     7141 cgtattagtc atcgctatta ccatg
//
