LOCUS       sequence-442508-        5621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adeno-associated viral vector to express HaloTag-GFP-mito fusion, a 
            cytosolic-localized Halotag-GFP fusion construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5621)
  AUTHORS   Deprey K, Batistatou N, Debets MF, Godfrey J, VanderWall KB, Miles 
            RR, Shehaj L, Guo J, Andreucci A, Kandasamy P, Lu G, Shimizu M, 
            Vargeese C, Kritzer JA
  TITLE     Quantitative Measurement of Cytosolic and Nuclear Penetration of 
            Oligonucleotide Therapeutics.
  JOURNAL   ACS Chem Biol. 2022 Jan 15. doi: 10.1021/acschembio.1c00830.
  PUBMED    35034446
REFERENCE   2  (bases 1 to 5621)
  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: "ACS Chem 
            Biol. 2022 Jan 15. doi: 10.1021/acschembio.1c00830."
COMMENT     Sequence Label: AAV-HGM (optimized)
FEATURES             Location/Qualifiers
     source          1..5621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   609..749
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   620..749
                     /label=AAV2 ITR
     protein_bind    756..776
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        837..1140
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1141..1344
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1295..1315
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1365..1389
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1390..1399
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1396..2286
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     regulatory      2293..2302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2299..2301,2302..2304,2305..3015)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2299..2301,2302..2304,2305..3015)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2344..2365)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2605..2624)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2952..2973
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3223..3247)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3271..3288)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3277..3484
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3492..3632
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3492..3621
                     /label=AAV2 ITR
     rep_origin      3707..4162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3794..3813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4004..4025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4179..4198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4298..4320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4358..4376)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4444..4548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4549..4617,4618..5409)
                     /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(4767..4786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5580..5621,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
      661 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      721 agtggccaac tccatcacta ggggttcctt ctagacaact ttgtatagaa aagttgtagt
      781 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      841 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      901 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      961 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1021 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1081 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1141 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1201 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1261 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1321 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccaagt ttgtacaaaa
     1381 aagcaggctg ccaccatggc agaaatcggt actggctttc cattcgaccc ccattatgtg
     1441 gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg
     1501 ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt
     1561 gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca
     1621 gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg
     1681 ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg
     1741 gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc
     1801 ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc
     1861 gacgtcggcc gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg
     1921 ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat
     1981 cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca
     2041 gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg
     2101 aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg
     2161 gccaaaagcc tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa
     2221 gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt
     2281 tccggttacg tagccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2341 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2401 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2461 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2521 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2581 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2641 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2701 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2761 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2821 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2881 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2941 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3001 gacgagctgt acaagggtgg tgaatccggt gatgcatcgg gaagtggaag tggatctcga
     3061 gctcaagctt cgaattcaaa actaattgct aaaagtgcag aagacgaaaa agcgaaggaa
     3121 gaaccaggga accataggat cgtaattctt gcaatgttag ctattggcgt gttctcttta
     3181 ggggcgctta tcaaaattat tcaattaaga aaaaataatt aaacccagct ttcttgtaca
     3241 aagtgggaat tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     3301 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3361 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3421 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     3481 gggagggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3541 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3601 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     3661 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3721 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3781 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3841 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3901 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     3961 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4021 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4081 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4141 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4201 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4261 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4321 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4381 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4441 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4501 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4561 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4621 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4681 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4741 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4801 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4861 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4921 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4981 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5041 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5101 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5161 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5221 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     5281 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5341 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5401 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5461 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5521 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5581 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa c
//
