LOCUS       sequence-420139-        6510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GABARAP split tandem protein with mTagBFP2, 5X: K2A, 
            K13A, R15A, K20A, K23A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6510)
  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 6510)
  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 GABARAP(5X/G116A)-mTagBFP2-GABARAP
FEATURES             Location/Qualifiers
     source          1..6510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     PPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2044..2065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2080..2085,2086..2103,2104..2110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2118..2134
                     /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)."
     primer_bind     2123..2145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2142..2158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2142..2158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2207..2328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2218..2237)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2272..2291
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2458..3480)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(2458..3480)
                     /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
                     "
     protein_bind    complement(3488..3535)
                     /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)."
     primer_bind     3667..3686
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3819..4043
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4032..4049
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4450..5160)
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(5618..5642)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5652..5670
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5673..5691
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(5693..5896)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5722..5742)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5897..6276
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6448..6467
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccctccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1921 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     1981 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2041 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     2101 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     2161 tgattacgcc aagctctagc tagaggtcga cggtatacag acatgataag atacattgat
     2221 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     2281 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa
     2341 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     2401 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gtacgtgcta
     2461 ttcctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac
     2521 acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc
     2581 ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt
     2641 gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag
     2701 ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat
     2761 acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa
     2821 catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt
     2881 ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat
     2941 cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca
     3001 gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc
     3061 gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat
     3121 cgcatccatg gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc
     3181 ttgcaacgtg acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctgaattc
     3241 cccaatgtca agcacttccg gaatcgggag cgcggccgat gcaaagtgcc gataaacata
     3301 acgatctttg tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc
     3361 tacatcgaag ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac
     3421 gctgtcgaac ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt
     3481 ggccaaggaa gttcctatac tttctagaga ataggaactt cggaatagga acttctaggt
     3541 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3601 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3661 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3721 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtggggata
     3781 ccccctagag ccccagctgg ttctttccgc ctcagaagcc atagagccca ccgcatcccc
     3841 agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc
     3901 cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag
     3961 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga
     4021 tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg gtttaagtgg gcccttacag
     4081 accgtagaca ctttcgtcac tgtaggcaat gtagagaaag aagtcttctt catggtgttc
     4141 ctggtacagc tgacccattg tggcactggt gggtggaatg acattgttga caaagaaaaa
     4201 caaggcatcc tcagctcgga gatgaattcg cttccggatc aagaagtaga actgaccaac
     4261 tgtgagatca gaaggcacca ggtatttctt tttgtccagg tctcctatcc gagctttggg
     4321 agccttttct actatcaccg gcacccggtc cgggtatttc tttcggatct tctcgccctc
     4381 agagcggcgc ttctcgaacg gatgctcttc tttgtacacg aacttcatag ctcgagatct
     4441 gagtccggaa ttaagcttgt gccccagttt gctagggagg tcgcagtatc tggccactgc
     4501 cacctcgtgc tgctcgacgt aggtctcgtt gttggcctcc ttgattcttt ccagtctgta
     4561 gtccacatag tagacgccag gcatcttgag gttcttagcg ggtttcttgg atctatatgt
     4621 ggtcttggcg tttgcgatca gatggctccc gcccacgagc ttcagggcca tgtcgtttct
     4681 gccttccagg ccgccgtcag cggggtacag cgtctcggtg aaggcctccc agccgagtgt
     4741 tttcttctgc atcacagggc cgttggatgt gaagttcacc cctctgatct tgacgttgta
     4801 gatgaggcag ccgtcctgga ggctggtgtc ctgggtagcg gtcagcacgc ccccgtcttc
     4861 gtatgtggtg actctctccc atgtgaagcc ctcagggaag gactgcttga agaagtcggg
     4921 gatgccctgg gtgtggttga tgaaggtctt gctgccgtag aggaagctag tagccaggat
     4981 gtcgaaggcg aaggggagag ggccgccctc gaccaccttg attctcatgg tctgggtgcc
     5041 ctcgtagggc ttgccttcgc cctcggatgt gcacttgaag tgatggttgt ccacggtgcc
     5101 ctccatgtac agcttcatgt gcatgttctc cttaatcagc tcttcgccct tagacaccat
     5161 ggatccatct gatccggaca gagcgtagac actttcgtca ctgtaggcaa tgtagagaaa
     5221 gaagtcttct tcatggtgtt cctggtacag ctgacccatt gtggcactgg tgggtggaat
     5281 gacattgttg acaaagaaaa acaaggcatc ctcagctcgg agatgaattc gcttccggat
     5341 caagaagtag aactgaccaa ctgtgagatc agaaggcacc aggtatttct ttttgtccag
     5401 gtctcctatc cgagctttgg gagccttttc tactatcacc ggcacccggt ccgggtattt
     5461 ggctcggatt gcctcgccct cagatgcgcg cgcctcgaac ggatgctctt ctttgtacac
     5521 gaatgccatg gttgcgttta aacgctagag tccggaggct ggatcggtcc cggtgtcttc
     5581 tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc actaaacgag
     5641 ctcgtcgacg atctctatca ctgataggga gatctctatc actgataggg agagctctgc
     5701 ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt
     5761 tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa
     5821 tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact
     5881 gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
     5941 gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     6001 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     6061 accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa
     6121 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     6181 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     6241 ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat
     6301 cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc
     6361 cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag
     6421 cagggagcag atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     6481 ataggggatc gggagatctc ccgatccgtc
//
