LOCUS       sequence-429630-        5621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adeno-associated viral vector expressing HaloTag-GFP-mito, a 
            cytosolically localized HaloTag-GFP fusion protein.
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
FEATURES             Location/Qualifiers
     source          1..5621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    529..553
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      554..563
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             560..1450
                     /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      1457..1466
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1463..1465,1466..1468,1469..2179)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1463..1465,1466..1468,1469..2179)
                     /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(1508..1529)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1769..1788)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2116..2137
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2387..2411)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2435..2452)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2441..2648
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2656..2796
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2656..2785
                     /label=AAV2 ITR
     rep_origin      2871..3326
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2958..2977)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3168..3189
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3343..3362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3462..3484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3522..3540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3608..3712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3713..3781,3782..4573)
                     /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(3931..3950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4744..5332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5233..5252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5394..5534
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5405..5534
                     /label=AAV2 ITR
     protein_bind    5541..5561
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc aagtttgtac
      541 aaaaaagcag gctgccacca tggcagaaat cggtactggc tttccattcg acccccatta
      601 tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc
      661 tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca
      721 tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa
      781 accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc
      841 cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca
      901 ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc
      961 tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac
     1021 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
     1081 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
     1141 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
     1201 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
     1261 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
     1321 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
     1381 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga
     1441 gatttccggt tacgtagcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     1501 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1561 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1621 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1681 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1741 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1801 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1861 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1921 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1981 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2041 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2101 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2161 catggacgag ctgtacaagg gtggtgaatc cggtgatgca tcgggaagtg gaagtggatc
     2221 tcgagctcaa gcttcgaatt caaaactaat tgctaaaagt gcagaagacg aaaaagcgaa
     2281 ggaagaacca gggaaccata ggatcgtaat tcttgcaatg ttagctattg gcgtgttctc
     2341 tttaggggcg cttatcaaaa ttattcaatt aagaaaaaat aattaaaccc agctttcttg
     2401 tacaaagtgg gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     2461 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2521 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2581 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     2641 gctggggagg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     2701 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     2761 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     2821 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     2881 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2941 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3001 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3061 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     3121 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3181 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     3241 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3301 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     3361 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3421 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3481 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     3541 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3601 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3661 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3721 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3781 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3841 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3901 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3961 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4021 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4081 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4141 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4201 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4261 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4321 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4381 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     4441 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4501 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4561 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4621 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4681 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4741 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4801 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4861 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4921 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4981 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5041 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5101 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5161 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5221 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5281 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5341 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     5401 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     5461 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     5521 cactaggggt tccttctaga caactttgta tagaaaagtt gtagttatta atagtaatca
     5581 attacggggt cattagttca tagcccatat atggagttcc g
//
