LOCUS       sequence-244033-        6440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-GABARAPL1 G116 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6440)
  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 6440)
  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. 
            2019 Jan 21. doi: 10.1080/15548627.2019.1569925."
COMMENT     Sequence Label: pDEST-CMV GFP-GABARAPL1 G116
FEATURES             Location/Qualifiers
     source          1..6440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             860..1576
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(860..862,863..865,866..1576)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(905..926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1166..1185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1513..1534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1598..1622
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1977..2001)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2033..2050)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2039..2263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2309..2737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2396..2415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2606..2627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2746..2766)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2751..3080
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2931..3066
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2993..3012
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3147..3941
                     /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(3201..3220)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3811..3830
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4115..4236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4152..4171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4206..4225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4285..4301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4285..4301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4298..4320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4309..4325
                     /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(4333..4339,4340..4357,4358..4363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4378..4399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4516..4533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4687..5275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4767..4786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5446..6237,6238..6306))
                     /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     6069..6088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6307..6411)
                     /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 accgtcagaa
      841 ttgatctggt accgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      901 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      961 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1021 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1081 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1141 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1201 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1261 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1321 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1381 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1441 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1501 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat
     1561 ggacgagctc tacaaaagcg gttccggacc ggatcaaaca agtttgtaca aaaaagcagg
     1621 cttaatgaag ttccagtaca aggaggacca tccctttgag tatcggaaaa aggaaggaga
     1681 aaagatccgg aagaaatatc cggacagggt ccccgtgatt gtagagaagg ctccaaaagc
     1741 cagggtgcct gatctggaca agaggaagta cctagtgccc tctgacctta ctgttggcca
     1801 gttctacttc ttaatccgga agagaatcca cctgagacct gaggacgcct tattcttctt
     1861 tgtcaacaac accatccctc ccaccagtgc taccatgggc caactgtatg aggacaatca
     1921 tgaggaagac tattttctgt atgtggccta cagtgatgag agtgtctatg ggtaataccc
     1981 agctttcttg tacaaagtgg tgataattaa ttaagataaa cccgctgatc agcctcgact
     2041 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2101 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2161 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2221 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2281 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2341 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2401 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2461 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2521 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2581 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2641 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2701 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     2761 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2821 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2881 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2941 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3001 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3061 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3121 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3181 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3241 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3301 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3361 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3421 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3481 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3541 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3601 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3661 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3721 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3781 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3841 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3901 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3961 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4021 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4081 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4141 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4201 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4261 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4321 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4381 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4441 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4501 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4561 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4621 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4681 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4741 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4801 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4861 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4921 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4981 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5041 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5101 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5161 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5221 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5281 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5341 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5401 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5461 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5521 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5581 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5641 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5701 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5761 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5821 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5881 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5941 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6001 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6061 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6121 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6181 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6241 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6301 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6361 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6421 ccgaaaagtg ccacctgacg
//
