LOCUS       sequence-421647-        6223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminally tagged GABARAP with eYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6223)
  AUTHORS   Uffing A, Gold L, Gensch T, Weiergraber OH, Hoffmann S, Willbold D
  TITLE     Highlighting the hidden: monitoring the avidity-driven association 
            of a fluorescent GABARAP tandem with microtubules in living cells.
  JOURNAL   Autophagy Rep. 2024 May 16;3(1):2348899. doi: 
            10.1080/27694127.2024.2348899. eCollection 2024.
  PUBMED    40395516
REFERENCE   2  (bases 1 to 6223)
  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 
            Rep."; date: "2024-05-16"; volume: "3(1):2348899. doi"; pages: " 
            10.1080/27694127.2024.2348899. eCollection 2024"
COMMENT     Sequence Label: pcDNA5/FRT/TO eYFP-GABARAP
FEATURES             Location/Qualifiers
     source          1..6223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      984..993
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(990..992,993..995,996..1706)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1035..1056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1296..1315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1643..1664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2175..2192)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2181..2405
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2538..2557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2689..2736
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2744..3766
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2744..3766
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3896..4017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3933..3952)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3987..4006
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4066..4082)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4066..4082)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4079..4101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4090..4106
                     /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(4114..4120,4121..4138,4139..4144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4159..4180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4297..4314)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4468..5056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4548..4567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5227..6018,6019..6087))
                     /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     5850..5869
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6088..6192)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttgccacca tggtgagcaa gggcgaggag ctgttcaccg
     1021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt
     1201 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1561 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag
     1621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1681 ctctcggcat ggacgagctg tacaagtccg gactcagatc tcgagctatg aagttcgtgt
     1741 acaaagaaga gcatccgttc gagaagcgcc gctctgaggg cgagaagatc cgaaagaaat
     1801 acccggaccg ggtgccggtg atagtagaaa aggctcccaa agctcggata ggagacctgg
     1861 acaaaaagaa atacctggtg ccttctgatc tcacagttgg tcagttctac ttcttgatcc
     1921 ggaagcgaat tcatctccga gctgaggatg ccttgttttt ctttgtcaac aatgtcattc
     1981 cacccaccag tgccacaatg ggtcagctgt accaggaaca ccatgaagaa gacttctttc
     2041 tctacattgc ctacagtgac gaaagtgtct acggtctgta aggatccact agtccagtgt
     2101 ggtggaattc tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta
     2161 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2221 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2281 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2341 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2401 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2461 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2521 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2581 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2641 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     2701 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     2761 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     2821 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     2881 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     2941 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     3001 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3061 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     3121 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     3181 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     3241 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3301 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3361 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3421 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     3481 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     3541 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     3601 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     3661 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     3721 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     3781 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3841 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     3901 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3961 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4021 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4081 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4141 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4201 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4261 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4321 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4381 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4441 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4501 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4561 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4621 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4681 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4741 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4801 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4861 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4921 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4981 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5041 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5101 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5161 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5221 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5281 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5341 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5401 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5461 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5521 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5581 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5641 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5701 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5761 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5821 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5881 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5941 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6001 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6061 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6121 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6181 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
