LOCUS       sequence-487264-        6655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of H2B-GFP for nuclei isolation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  TITLE     Maria J Galazo Lab Materials
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6655)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAV-CAG-H2B-GFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        542..819
                     /label=chicken beta-actin promoter
     intron          820..1836
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1760..1782
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1844..1863
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1890..1914
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1921..2298
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             join(2317..2319,2320..3033)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2317..3030
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2347..2375)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2970..2990
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(3034..3058)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3068..3656
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3121..3141)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3539..3550)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3686..3703)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3692..3899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3907..4047
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3907..4036
                     /label=AAV2 ITR
     rep_origin      4122..4577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4209..4228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4419..4440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4594..4613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4713..4735
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4773..4791)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4859..4963
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4964..5032,5033..5824)
                     /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(5182..5201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5995..6583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6484..6503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6645..6655,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgctc gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
      601 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
      661 gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
      721 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      781 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      841 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      901 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
      961 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
     1021 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
     1081 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
     1141 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
     1201 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
     1261 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
     1321 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
     1381 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag
     1441 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
     1501 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
     1561 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
     1621 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     1681 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     1741 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     1801 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     1861 gtgctgtctc atcattttgg caaagaattg caagtttgta caaaaaagca ggctgccacc
     1921 atgccagagc cagcgaagtc tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact
     1981 aaggcgcaga agaaaggcgg caagaagcgc aagcgcagcc gcaaggagag ctattccatc
     2041 tatgtgtaca aggttctgaa gcaggtccac cctgacaccg gcatttcgtc caaggccatg
     2101 ggcatcatga attcgtttgt gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc
     2161 ctggcgcatt acaacaagcg ctcgaccatc acctccaggg agatccagac ggccgtgcgc
     2221 ctgctgctgc ctggggagtt ggccaagcac gccgtgtccg agggtactaa ggccgtcacc
     2281 aagtacacca gcgctaagga cccaccggtc gccaccatga gcaaaggaga agaacttttc
     2341 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
     2401 gtccgtggag agggtgaagg tgatgctaca aacggaaaac tcacccttaa atttatttgc
     2461 actactggaa aactacctgt tccatggcca acacttgtca ctactctgac ctatggtgtt
     2521 caatgctttt cccgttatcc ggatcacatg aaacggcatg actttttcaa gagtgccatg
     2581 cccgaaggtt atgtacagga acgcactata tctttcaaag atgacgggac ctacaagacg
     2641 cgtgctgaag tcaagtttga aggtgatacc cttgttaatc gtatcgagtt aaaaggtatt
     2701 gattttaaag aagatggaaa cattctcgga cacaaactcg agtacaactt taactcacac
     2761 aatgtataca tcacggcaga caaacaaaag aatggaatca aagctaactt caaaattcgc
     2821 cacaacgttg aagatggttc cgttcaacta gcagaccatt atcaacaaaa tactccaatt
     2881 ggcgatggcc ctgtcctttt accagacaac cattacctgt cgacacaatc tgtcctttcg
     2941 aaagatccca acgaaaagcg tgaccacatg gtccttcttg agtttgtaac tgctgctggg
     3001 attacacatg gcatggatga gctctacaaa taaacccagc tttcttgtac aaagtgggaa
     3061 ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3121 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3181 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3241 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3301 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3361 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3421 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     3481 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3541 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3601 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     3661 ggaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3721 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3781 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3841 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     3901 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     3961 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4021 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4081 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4141 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4201 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4261 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4321 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4381 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4441 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4501 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4561 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4621 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4681 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4741 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4801 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4861 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4921 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4981 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5041 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5101 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5161 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5221 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5281 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5341 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5401 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5461 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5521 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5581 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5641 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     5701 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5761 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5821 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5881 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5941 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6001 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6061 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6121 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6181 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6241 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6301 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6361 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6421 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6481 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6541 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6601 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcag
//
