LOCUS       sequence-472362-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CAGGS driven nls BFP with a FLAG tag followed by a WPRE-bGH polyA 
            for stable transcripts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 6630)
  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: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pCAGEN nls-BFP-FLAG-WPRE-bgh-pA (JDW 452)
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1745..1769
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1778..1787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1811..1831
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1835..1855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1859..1879
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1895..2593
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    2594..2659
                     /label=MCS
                     /note="multiple cloning site"
     CDS             2672..2695
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2765..2785)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3183..3194)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3303..3319)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3304..3320)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3321..3338)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3327..3438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3536..3560)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3652..3671)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3717..3772
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3771..3790)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4133..4149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4133..4149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4146..4168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4157..4173
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4181..4187,4188..4205,4206..4211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4226..4247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4306..4501
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4352..4487
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4507..4641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4544..4563)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4598..4617
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4709..4726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4880..5468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4960..4979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5639..6430,6431..6499))
                     /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     6262..6281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6500..6604)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt atttggggga
      481 gggggggggg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagga
     1741 tatcacaagt ttgtacaaaa aagcaggctg cgatcgcgcc accatggcca caaccatggt
     1801 gcacgtggat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa aggtagatcc
     1861 aaaaaagaag agaaaggtac acgtgtccac gcgtatgagc gagctgatta aggagaacat
     1921 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     1981 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     2041 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     2101 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     2161 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     2221 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     2281 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     2341 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     2401 tctgatcgca aacatcaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     2461 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     2521 ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg
     2581 gcacaagctt aattccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg
     2641 taccgcgggc ccgggatcca ccggatctag agactacaaa gacgatgacg acaagtagag
     2701 ggagctcatc tagtcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2761 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2821 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2881 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2941 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3001 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3061 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3121 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3181 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3241 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3301 atcgataccg tcgacctcga cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3361 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3421 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3481 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatggcg atcgcaccca
     3541 gctttcttgt acaaagtggt gatatcgcgg ccgcactcct caggtgcagg ctgcctatca
     3601 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     3661 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     3721 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3781 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3841 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     3901 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     3961 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     4021 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     4081 tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc
     4141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc
     4321 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4381 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4441 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4501 aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4561 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4621 gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4741 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4801 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4861 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4921 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4981 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5041 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5101 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5161 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5221 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5281 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5341 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5401 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5461 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5521 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5581 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5641 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5701 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5761 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5821 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5881 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5941 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6001 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6061 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6121 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6181 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6241 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6301 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6361 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6421 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6481 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6541 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6601 cgcgcacatt tccccgaaaa gtgccacctg
//
