LOCUS       sequence-96169-e        6684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses membrane-targeted SNAP(f)-tag and histone-GFP, 
            conditional.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6684)
  AUTHORS   Kohl J, Ng J, Cachero S, Ciabatti E, Dolan MJ, Sutcliffe B, Tozer A,
            Ruehle S, Krueger D, Frechter S, Branco T, Tripodi M, Jefferis GS
  TITLE     Ultrafast tissue staining with chemical tags.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Aug 25. pii: 201411087.
  PUBMED    25157152
REFERENCE   2  (bases 1 to 6684)
  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: "Proc Natl 
            Acad Sci U S A. 2014 Aug 25. pii: 201411087."
COMMENT     Sequence Label: pAAV-myrSNAP-CON
FEATURES             Location/Qualifiers
     source          1..6684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        221..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        525..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     678..698
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..748
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          876..1254
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     protein_bind    complement(1332..1365)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1436..2146,2147..2149))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1478..1499)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1827..1846
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2086..2107
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2234..2611)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMSSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             complement(2630..2683)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2693..3238)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     regulatory      3298..3307
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3361..3394
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3431..3907
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3630..3649)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3947..4087
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3947..4076
                     /label=AAV2 ITR
     rep_origin      4162..4617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4249..4268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4459..4480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4634..4653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4753..4775
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4813..4831)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4899..5003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5004..5072,5073..5864)
                     /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(5222..5241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6035..6623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6524..6543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtggagc tagttattaa tagtaatcaa
      181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      301 ttcccatagt aacgtcaata gggactttcc attgacgtca atgggtggag tatttacggt
      361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      601 ttgacgtcaa tgggagtttg ttttgcacca aaatcaacgg gactttccaa aatgtcgtaa
      661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      721 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      781 ccatagaaga caccgggacc gatccagcct ccgcggattc gaatcccggc cgggaacggt
      841 gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata
      901 ggcccacaaa aaatgctttc ttcttttaat atactttttt gtttatctta tttctaatac
      961 tttccctaat ctctttcttt cagggcaata atgatacaat gtatcatgcc tctttgcacc
     1021 attctaaaga ataacagtga taatttctgg gttaaggcaa tagcaatatt tctgcatata
     1081 aatatttctg catataaatt gtaactgatg taagaggttt catattgcta atagcagcta
     1141 caatccagct accattctgc ttttatttta tggttgggat aaggctggat tattctgagt
     1201 ccaagctagg cccttttgct aatcatgttc atacctctta tcttcctccc acagctcctg
     1261 ggcaacgtgc tggtctgtgt gctggcccat cactttggca aagaattggg attcgaacat
     1321 cgattgaatt cataacttcg tatagcatac attatacgaa cggtatcact attacttact
     1381 atcacctatt atcacggccg cccggatccg atgagcttga tgatctgagt ccggacttgt
     1441 acagctcgtc catgccgtga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     1501 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt
     1561 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
     1621 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt ggccttgatg ccgttcttct
     1681 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     1741 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1801 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1861 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1921 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     1981 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     2041 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     2101 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcatg gtggcccgcg
     2161 gtagcgctag cccacctccg ccgctgcccc cacctccaga tccacccccg ccggagcctc
     2221 cacccccaga tcccttagcg ctggtgtact tggtgacggc cttagtaccc tcggacacgg
     2281 cgtgcttggc caactcccca ggcagcagca ggcgcacggc cgtctggatc tccctggagg
     2341 tgatggtcga gcgcttgttg taatgcgcca ggcgggaagc ctcacctgcg atgcgctcga
     2401 aaatgtcgtt cacaaacgaa ctcatgatgc ccatggcctt ggacgaaatg ccggtgtcag
     2461 ggtggacctg cttcagaacc ttgtacacat agatggaata gctctccttg cggctgcgct
     2521 tgcgcttctt gccgcctttc ttctgcgcct tagtcaccgc cttcttggag ccctttttcg
     2581 gggcgggagc agacttcgct ggctctggca tggtgatatt cagatctcct gggccaggat
     2641 tctcctcgac gtcaccgcat gttagcagac ttcctctgcc ctctccactg ccacccagcc
     2701 caggcttgcc cagtctgtgg ccctcgtggg ccagcagcca ctctttcacg gcgagcccgc
     2761 cctcgtagcc ccccacgtcc aggtcgccct gcaccacccg gtggcagggg atcagaatgg
     2821 gcacgggatt tccgctcagg gcggttttca cggcggcggt ggcggcggga ttgccggcca
     2881 gggcggccag gtggctgtag ctgatgacct ctccgaactt caccactttc agcagtttcc
     2941 acagcacctg gcgggtaaag ctctcctgct ggaacactgg gtggtgcagg gctggcacag
     3001 ggaactcctc gatggcctca ggctggtgaa agtaggcgtt gagccaggcg gtggcctgca
     3061 tcagtggctc tggtccgccc agcacggcgg ctggggcagg cacttccacg gcgtcggcgg
     3121 cagatgttcc tttgcccagg aagatgatac ggtgcaggcc ctgttcgcac ccagacagtt
     3181 ccagcttgcc cagagggcta tccagggtgg tgcgcttcat ttcgcagtct ttgtccatgg
     3241 aggcgaccgg tgcctcgtcg tcgttcaggt tgtccttgcg cttgctcttg atgcagccca
     3301 tggcggcctc gtcgtcgttc aggttgtcct tgcgcttgct cttgatgcag cccatggtgg
     3361 ataacttcgt ataatgtatg ctatacgaac ggtaaagctt gcctcgagca gcgctgctcg
     3421 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     3481 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     3541 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     3601 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     3661 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     3721 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     3781 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     3841 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     3901 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     3961 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4021 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4081 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4141 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4501 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4561 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     4621 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4681 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4741 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4801 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4861 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4921 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4981 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5041 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5101 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5161 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5221 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5281 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5341 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5401 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5461 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5521 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5581 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5641 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5701 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5761 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5821 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5881 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5941 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6001 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6061 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6181 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6661 tttgctggcc ttttgctcac atgt
//
