LOCUS       sequence-370452-        8176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adeno-associated viral vector to express histone2B-HaloTag-GFP 
            fusion, a nuclear-localized Halotag-GFP fusion construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8176)
  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 8176)
  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-H2GH
FEATURES             Location/Qualifiers
     source          1..8176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     590..614
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          707..1182
                     /label=beta-globin intron
                     /note="internally truncated intron from human beta-globin"
     CDS             1259..1636
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1649..1658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1655..1657,1658..1660,1661..2371)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1655..1657,1658..1660,1661..2371)
                     /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(1700..1721)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1961..1980)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2308..2329
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2375..2384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2381..3268
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEIS"
     primer_bind     complement(3300..3316)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3301..3317)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3410..3804
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     repeat_region   4159..4288
                     /label=AAV2 ITR
     rep_origin      4659..5172
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4746..4765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4956..4977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5654..5673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5773..5795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5833..5851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5919..6023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6024..6092,6093..6884)
                     /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(6242..6261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7055..7643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7544..7563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7880..8009
                     /label=AAV2 ITR
     primer_bind     complement(8065..8084)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
      601 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      661 gggaccgatc cagcctcccc tcgaagctga tcctgagaac ttcagggtga gtctatggga
      721 cccttgatgt tttctttccc cttcttttct atggttaagt tcatgtcata ggaaggggag
      781 aagtaacagg gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa
      841 atgctttctt cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct
      901 ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat
      961 aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca
     1021 tataaattgt aactgatgta agaggtttca tattgctaat agcagctaca atccagctac
     1081 cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc
     1141 cttttgctaa tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg
     1201 gtctgtgtgc tggcccatca ctttggcaaa gaattccgcg ggcccgggat ccaccggtat
     1261 gccagagcca gcgaagtctg ctcccgcccc gaaaaagggc tccaagaagg cggtgactaa
     1321 ggcgcagaag aaaggcggca agaagcgcaa gcgcagccgc aaggagagct attccatcta
     1381 tgtgtacaag gttctgaagc aggtccaccc tgacaccggc atttcgtcca aggccatggg
     1441 catcatgaat tcgtttgtga acgacatttt cgagcgcatc gcaggtgagg cttcccgcct
     1501 ggcgcattac aacaagcgct cgaccatcac ctccagggag atccagacgg ccgtgcgcct
     1561 gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag ggtactaagg ccatcaccaa
     1621 gtacaccagc gctaaggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt
     1681 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1741 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1801 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1861 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1921 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1981 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2041 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2101 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2161 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2221 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2281 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2341 gatcactctc ggcatggacg agctgtacaa gcgcgccacc atggcagaaa tcggtactgg
     2401 ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt
     2461 tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt
     2521 gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat
     2581 cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt
     2641 catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg
     2701 gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc
     2761 atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga
     2821 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
     2881 ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga
     2941 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
     3001 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
     3061 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
     3121 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat
     3181 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     3241 gcgctggctg tcgacgctcg agatttccta agcggccgct ctagaggatc caagcttatc
     3301 gataccgtcg acctcgaggg cccagatcta attcacccca ccagtgcagg ctgcctatca
     3361 gaaagtggtg gctggtgtgg ctaatgccct ggcccacaag tatcactaag ctcgctttct
     3421 tgctgtccaa tttctattaa aggttccttt gttccctaag tccaactact aaactggggg
     3481 atattatgaa gggccttgag catctggatt ctgcctaata aaaaacattt attttcattg
     3541 caatgatgta tttaaattat ttctgaatat tttactaaaa agggaatgtg ggaggtcagt
     3601 gcatttaaaa cataaagaaa tgaagagcta gttcaaacct tgggaaaata cactatatct
     3661 taaactccat gaaagaaggt gaggctgcaa acagctaatg cacattggca acagcccctg
     3721 atgcctatgc cttattcatc cctcagaaaa ggattcaagt agaggcttga tttggaggtt
     3781 aaagttttgc tatgctgtat tttacattac ttattgtttt agctgtcctc atgaatgtct
     3841 tttcactacc catttgctta tcctgcatct ctcagccttg actccactca gttctcttgc
     3901 ttagagatac cacctttccc ctgaagtgtt ccttccatgt tttacggcga gatggtttct
     3961 cctcgcctgg ccactcagcc ttagttgtct ctgttgtctt atagaggtct acttgaagaa
     4021 ggaaaaacag ggggcatggt ttgactgtcc tgtgagccct tcttccctgc ctcccccact
     4081 cacagtgacc cggaatccct cgacatctag agcatggcta cgtagataag tagcatggcg
     4141 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4201 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4261 cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc cgcaccgatc
     4321 gcccttccca acagttgcgc agcctgaatg gcgaatggcg attccgttgc aatggctggc
     4381 ggtaatattg ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca
     4441 agtgatgtta ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggacag
     4501 actcttttac tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg
     4561 ttcctgtcta aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag
     4621 gaaagcacgt tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4681 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4741 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4801 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4861 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4921 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4981 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5041 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5101 aacgcttaca atttaaatat ttgcttatac aatcttcctg tttttggggc ttttctgatt
     5161 atcaaccggg gtacatatga ttgacatgct agttttacga ttaccgttca tcgattctct
     5221 tgtttgctcc agactctcag gcaatgacct gatagccttt gtagagacct ctcaaaaata
     5281 gctaccctct ccggcatgaa tttatcagct agaacggttg aatatcatat tgatggtgat
     5341 ttgactgtct ccggcctttc tcacccgttt gaatctttac ctacacatta ctcaggcatt
     5401 gcatttaaaa tatatgaggg ttctaaaaat ttttatcctt gcgttgaaat aaaggcttct
     5461 cccgcaaaag tattacaggg tcataatgtt tttggtacaa ccgatttagc tttatgctct
     5521 gaggctttat tgcttaattt tgctaattct ttgccttgcc tgtatgattt attggatgtt
     5581 ggaatcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     5641 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5701 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5761 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5821 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5881 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6001 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6061 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6121 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6181 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6241 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6301 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6361 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6421 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6481 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6541 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6601 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6661 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6721 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6781 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6841 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6901 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6961 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7021 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     7081 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     7141 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7201 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7261 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7321 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7381 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7441 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7501 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7561 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7621 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7681 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7741 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     7801 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     7861 ggccgattca ttaatgcagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
     7921 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     7981 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
     8041 atctacgtag ccatgctcta gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     8101 catgagcttg gcccattgca tacgttgtat ccatatcata atatgtacat ttatattggc
     8161 tcatgtccaa cattac
//
