LOCUS       sequence-478589-        7259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HA-tagged human GABARAP in mammalian cells. Stable 
            expression with piggybac transposase. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7259)
  AUTHORS   Bentley-DeSousa A, Roczniak-Ferguson A, Ferguson SM
  TITLE     A STING-CASM-GABARAP pathway activates LRRK2 at lysosomes.
  JOURNAL   J Cell Biol. 2025 Feb 3;224(2):e202310150. doi: 
            10.1083/jcb.202310150. Epub 2025 Jan 15.
  PUBMED    39812709
REFERENCE   2  (bases 1 to 7259)
  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 Cell 
            Biol."; date: "2025-02-3"; volume: "224(2):e202310150. doi"; pages: 
            " 10.1083/jcb.202310150. Epub 2025 Jan 15"
COMMENT     Sequence Label: pPB-HA-hGABARAP
FEATURES             Location/Qualifiers
     source          1..7259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    316..336
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        337..1515
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          568..1506
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1463..1483
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1515..1539
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1549..1575
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1948..1972)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2050..2069)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2115..2170
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2169..2188)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        2525..2904
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2905..3108
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3059..3079
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3135..3144
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3141..3539
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3577..3594)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3583..3807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4157..4219
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4285..4304)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4286..4304)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4311..4328)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4311..4327)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4320..4342)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4469..4924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4556..4575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4766..4787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4950..5054
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5055..5123,5124..5915)
                     /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     complement(5273..5292)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6086..6674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6575..6594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6828..6845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6962..6983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6998..7003,7004..7021,7022..7028)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7036..7052
                     /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     7041..7063
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7060..7076
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7060..7076
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7097..7115
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat atcatcaact ttgtatagaa aagttgggct ccggtgcccg tcagtgggca
      361 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
      421 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      481 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      541 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
      601 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
      661 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
      721 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
      781 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
      841 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
      901 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
      961 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     1021 gggtagtctc aagctggccg gcctgctctg gtgcctggtc tcgcgccgcc gtgtatcgcc
     1081 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     1141 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     1201 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     1261 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     1321 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     1381 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     1441 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     1501 tttcaggtgt cgtgacaagt ttgtacaaaa aagcaggctg ccaccatgta cccatacgat
     1561 gttccagatt acgctggcag cggcagcggc agcatgaagt tcgtgtacaa agaagagcat
     1621 ccgttcgaga agcgccgctc tgagggcgag aagatccgaa agaaataccc ggaccgggtg
     1681 ccggtgatag tagaaaaggc tcccaaagct cggataggag acctggacaa aaagaaatac
     1741 ctggtgcctt ctgatctcac agttggtcag ttctacttct tgatccggaa gcgaattcat
     1801 ctccgagctg aggatgcctt gtttttcttt gtcaacaatg tcattccacc caccagtgcc
     1861 acaatgggtc agctgtacca ggaacaccat gaagaagact tctttctcta cattgcctac
     1921 agtgacgaaa gtgtctacgg tctgtgaacc cagctttctt gtacaaagtg gtgatcctca
     1981 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     2041 ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     2101 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     2161 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     2221 tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct
     2281 ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa
     2341 agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac
     2401 atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact
     2461 cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagcttcg
     2521 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2581 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2641 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2701 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2761 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     2821 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     2881 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     2941 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     3001 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3061 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     3121 agagaaccca ctgcgccacc atggccaagc ctttgtctca agaagaatcc accctcattg
     3181 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
     3241 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
     3301 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
     3361 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
     3421 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
     3481 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac
     3541 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3601 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3661 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3721 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3781 atgctgggga tgcggtgggc tctatggctc gagttaatta acgagagcat aatattgata
     3841 tgtgccaaag ttgtttctga ctgactaata agtataattt gtttctatta tgtataggtt
     3901 aagctaatta cttattttat aatacaacat gactgttttt aaagtacaaa ataagtttat
     3961 ttttgtaaaa gagagaatgt ttaaaagttt tgttacttta tagaagaaat tttgagtttt
     4021 tgtttttttt taataaataa ataaacataa ataaattgtt tgttgaattt attattagta
     4081 tgtaagtgta aatataataa aacttaatat ctattcaaat taataaataa acctcgatat
     4141 acagaccgat aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa
     4201 tatgattatc tttctagggt taaataatag tttctaattt ttttattatt cagcctgctg
     4261 tcgtgaatac cgagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc
     4321 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4381 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4441 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4501 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4561 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4621 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4681 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4741 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4801 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4861 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4921 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4981 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5041 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5101 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5161 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5221 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5281 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5341 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5401 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5461 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5521 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5581 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5641 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5701 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5761 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5821 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5881 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5941 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6001 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6061 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6241 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6841 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6901 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6961 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     7021 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     7081 attacgccaa gctcgaaatt aaccctcact aaagggaaca aaagctggta cctcgcgcga
     7141 cttggtttgc cattctttag cgcgcgtcgc gtcacacagc ttggccacaa tgtggttttt
     7201 gtcaaacgaa gattctatga cgtgtttaaa gtttaggtcg agtaaagcgc aaatctttt
//
