LOCUS       sequence-383153-        6481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  to express nuclear localized eBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6481)
  AUTHORS   Back F, Sandoval A, Vu LM, Hong VM, Bhaskara A, Rodriguez SR, 
            O'Brien JT, Kolber BJ, Kroener S, Ploski JE
  TITLE     Adeno-associated viral vector resource for the RNA-targeting Cas13d:
            A comparison of high-fidelity variants, DjCas13d and hfCas13d.
  JOURNAL   Mol Ther Methods Clin Dev. 2025 Aug 20;33(4):101565. doi: 
            10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11.
  PUBMED    41020266
REFERENCE   2  (bases 1 to 6481)
  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: "Mol Ther 
            Methods Clin Dev."; date: "2025-08-20"; volume: "33(4):101565. doi";
            pages: " 10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11"
COMMENT     Sequence Label: pAAV-2X(CMV-eBFP2-3XNLS-pA)
FEATURES             Location/Qualifiers
     source          1..6481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        980..1283
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1284..1487
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      1526..1535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1532..1534,1535..1537,1538..2248)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             2273..2293
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2297..2317
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2321..2341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2471..2592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        2712..3015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3016..3219
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3258..3267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3264..3266,3267..3269,3270..3980)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             4005..4025
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4029..4049
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4053..4073
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4203..4324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      4609..5064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4696..4715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4906..4927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5081..5100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5200..5222
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5260..5278)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5346..5450
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5451..5519,5520..6311)
                     /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(5669..5688)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcaattc agtcgataac tataacggtc ctaaggtagc gatttaaata cgcgctctct
      841 taaggtagcc ccgggacgcg tcaattgaga tctggatccg gtacctgatt ctgtggataa
      901 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
      961 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1021 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1081 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1141 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1201 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1261 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1321 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1381 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1441 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
     1501 gtcagatccg ctagcgctac cggtcgccac catggtgagc aagggcgagg agctgttcac
     1561 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1621 gaggggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
     1681 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgagcc acggcgtgca
     1741 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1801 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcacct acaagacccg
     1861 cgccgaggtg aagttcgagg gcgacaccct agtgaaccgc atcgagctga agggcgtcga
     1921 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
     1981 catctatatc atggccgtca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2041 caacgtggag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2101 cgacggcccc gtgctgctgc ccgacagcca ctacctgagc acccagtccg tgctgagcaa
     2161 agaccccaac gagaagcgcg atcacatggt cctgctggag ttccgcaccg ccgccgggat
     2221 cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagctg atccaaaaaa
     2281 gaagagaaag gtagatccaa aaaagaagag aaaggtagat ccaaaaaaga agagaaaggt
     2341 aggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     2401 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2461 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2521 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2581 caatgtatct taaggcgtaa attgtaagcg cggccgctga ttctgtggat aaccgtatta
     2641 ccgccatgca ttagttatta atagtaatca attacggggt cattagttca tagcccatat
     2701 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     2761 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     2821 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2881 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2941 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     3001 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     3061 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     3121 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     3181 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
     3241 cgctagcgct accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg
     3301 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgaggggcg
     3361 agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca
     3421 agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtg cagtgcttcg
     3481 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3541 acgtccagga gcgcaccatc ttcttcaagg acgacggcac ctacaagacc cgcgccgagg
     3601 tgaagttcga gggcgacacc ctagtgaacc gcatcgagct gaagggcgtc gacttcaagg
     3661 aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacatctata
     3721 tcatggccgt caagcagaag aacggcatca aggtgaactt caagatccgc cacaacgtgg
     3781 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3841 ccgtgctgct gcccgacagc cactacctga gcacccagtc cgtgctgagc aaagacccca
     3901 acgagaagcg cgatcacatg gtcctgctgg agttccgcac cgccgccggg atcactctcg
     3961 gcatggacga gctgtacaag tccggactca gatctcgagc tgatccaaaa aagaagagaa
     4021 aggtagatcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtaggatcca
     4081 ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     4141 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     4201 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4261 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4321 cttaaggcgt aaattgtaag caagcttcta gatatcctct cttaaggtag catcgagatt
     4381 taaattaggg ataacagggt aatggcgcgg gccgcaggaa cccctagtga tggagttggc
     4441 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccggcc
     4501 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4561 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4621 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4681 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4741 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4801 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4861 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4921 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4981 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5041 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5101 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5161 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5221 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5281 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5341 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5401 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5461 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5521 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5581 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5641 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5701 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5761 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5821 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5881 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5941 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6001 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6061 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6121 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6181 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6241 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6301 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6361 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6421 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6481 c
//
