LOCUS       sequence-496512-        7463 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Overexpression of BFP. Contains Cp36 recombination site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  AUTHORS   Arana S, Du PP, Vaughan-Jackson A, Enright N, Spees K, Valbuena R, 
            Garcia CA, Nguyen T, Venida A, Seczynska M, Bintu L, Lehner PJ, 
            Prolo LM, Bassik MC
  TITLE     Reduced Cas9 transgene silencing by incorporation of intron 
            sequences.
  JOURNAL   Nat Commun. 2025 Nov 27;16(1):10656. doi: 
            10.1038/s41467-025-65669-0.
  PUBMED    41309578
REFERENCE   2  (bases 1 to 7463)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-11-27"; volume: "16(1):10656. doi"; pages: " 
            10.1038/s41467-025-65669-0"
COMMENT     Sequence Label: pSA081 BFP
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        500..1709
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1546..1565
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             1713..2111
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2138..2186
                     /note="synthetic polyadenylation signal"
     CDS             join(2452..2520,2521..3312)
                     /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(2670..2689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      3325..3461
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3613..3684
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(3689..3735)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3753..3801
                     /note="synthetic polyadenylation signal"
     promoter        3942..5120
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4173..5111
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5068..5088
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5332..6033
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6085..6206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6096..6115)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6150..6169
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      6321..6367
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      6397..6533
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6578..6649
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(6875..7463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6955..6974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt
      241 aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat
      301 cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc
      361 tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga
      421 tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata
      481 tcgaaatcgg ggcgcgcctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc
      541 ctcgtcacgg cgagcgctgc cacgtcagac gaagggcgca ggagcgtcct gatccttccg
      601 cccggacgct caggacagcg gcccgctgct cataagactc ggccttagaa ccccagtatc
      661 agcagaagga cattttagga cgggacttgg gtgactctag ggcactggtt ttctttccag
      721 agagcggaac aggcgaggaa aagtagtccc ttctcggcga ttctgcggag ggatctccgt
      781 ggggcggtga acgccgatga ttatataagg acgcgccggg tgtggcacag ctagttccgt
      841 cgcagccggg atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga
      901 gtagcgggct gctgggctgg ccggggcttt cgtggccgcc gggccgctcg gtgggacgga
      961 agcgtgtgga gagaccgcca agggctgtag tctgggtccg cgagcaaggt tgccctgaac
     1021 tgggggttgg ggggagcgca gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc
     1081 ttgtgaggcg ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg cggcaagaac
     1141 ccaaggtctt gagcccttcg ctaatgcggg aaagctctta ttcgggtgag atgggctggg
     1201 caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct
     1261 gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg
     1321 cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc
     1381 acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
     1441 ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
     1501 gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt
     1561 gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa
     1621 tgtaatcatt tgggtcaata tgtaattttc agtgttagac ttgtaaattg tccgctaaat
     1681 tctggccgtt tttggctttt ttgttagaca acatggccaa gcctttgtct caagaagaat
     1741 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     1801 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     1861 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     1921 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     1981 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     2041 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     2101 gggagggcta agctagtatg taagcctagt cttagataat aaaatcgcta tccatcgaag
     2161 atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg
     2221 atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc cagtcggaat
     2281 ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg gtcacggcag
     2341 cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta taatcgagtc
     2401 ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg catgagtatt
     2461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc gcgagtgggt
     2581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgc
     2641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtat
     2761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat tggaggaccg
     2881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3001 atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3061 cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3121 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3181 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3301 aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg
     3361 cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc
     3421 cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcccgaa tcgtttaaac
     3481 tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc
     3541 atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt tggcagcgat
     3601 cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct ctagtggtca
     3661 taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt ttagaggccc
     3721 caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac acatccatct
     3781 tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccctta accgcttttg
     3841 aatgtccgtc ttagttaatc cctaattgaa cgctccaaaa ggtgacttcc aatagggatt
     3901 tattcctttt aaaattaacg gcataatcgt agatctcgtg aggctccggt gcccgtcagt
     3961 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     4021 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     4081 gcctttttcc cgagggtggg ggagaaccgt atataagtgc actagtcgcc gtgaacgttc
     4141 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     4201 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     4261 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg tggccttgcg
     4321 cttaaggagc cccttcgcct cgtgcttgag ttgtggcctg gcctgggcgc tggggccgcc
     4381 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     4441 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     4501 cgggccaaga tcagcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     4561 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     4621 gacgggggta gtctcaagct gcccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     4681 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     4741 ggccgcttcc cggccctgct gcagggagca caaaatggag gacgcggcgc tcgggagagc
     4801 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     4861 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcc agcttttgga
     4921 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     4981 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     5041 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     5101 ttccatttca ggtgtcgtga aaactacccc taaaagccaa aagatctttg tcgatcctac
     5161 catccactcg acacacccgc cagcggccgc tgccaagctt gctaggaatt cgtaacgctc
     5221 ttcgagaaag cagattctca tatccatctt gagtcttctt tctcgcaaga caacacgaaa
     5281 tagacacagt ctcttcccag ctgtacactg agccgaattc ggtacgccac catgagcgag
     5341 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     5401 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     5461 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     5521 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     5581 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     5641 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     5701 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     5761 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     5821 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc
     5881 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     5941 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     6001 ctccctagca aactggggca caagcttaat tagtaactag cttgactgac tgagatacag
     6061 cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa accacaacta
     6121 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     6181 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     6241 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg
     6301 gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg tcttgagggg
     6361 ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa aaaaatgcct
     6421 gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt
     6481 ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct taaggtcgtc
     6541 agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg gtcttttctg
     6601 gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa tctcattggt
     6661 taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg agatcgactt
     6721 gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt ccttgctatt
     6781 gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg
     6841 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6901 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6961 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     7021 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     7081 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     7141 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     7201 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     7261 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     7321 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7381 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7441 cgcgcagaaa aaaaggatct caa
//
