LOCUS       sequence-477259-        8621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosolic expression of mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8621)
  AUTHORS   Goldman, C., Kareva, T., Sarrafha, L., Schuldt, B.R., Sahasrabudhe, 
            A., Ahfeldt, T.,, Blanchard, J.W
  TITLE     Genetically encoded and modular subcellular organelle probes reveal 
            dysfunction in lysosomes and mitochondria driven by PRKN knockout
  JOURNAL   iScience 28 (2025)
REFERENCE   2  (bases 1 to 8621)
  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: "iScience 28
            (2025)"
COMMENT     Sequence Label: pFUW mTagBFP2-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             836..1016
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1063..1188
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1681..1914
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2099..2143
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2441..2558
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2623..3834
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3671..3690
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3865..3874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3871..4581
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             4606..4662
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4663..5262
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4663..4682)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5159..5179
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5288..5876
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5341..5361)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5759..5770)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5879..5895)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5880..5896)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6401..6581
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6607..6624)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6613..6724
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(6870..7458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6950..6969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7629..8420,8421..8489))
                     /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     8252..8271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8490..8594)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      841 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      901 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      961 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1021 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1081 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1141 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1201 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1261 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1321 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1381 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1441 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1501 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1561 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1621 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1681 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1741 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1801 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1861 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1921 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1981 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     2041 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     2101 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     2161 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     2221 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     2281 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga
     2341 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg
     2401 tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2461 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2521 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2581 gagatccagt ttggttaatt aacccgtgtc ggctccagat ctggcctccg cgccgggttt
     2641 tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc tgccacgtca gacgaagggc
     2701 gcagcgagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag
     2761 actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact
     2821 ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg
     2881 gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat aaggacgcgc
     2941 cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg
     3001 atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc
     3061 cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct gtagtctggg
     3121 tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa tggcggctgt
     3181 tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg
     3241 gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc ttcgctaatg cgggaaagct
     3301 cttattcggg tgagatgggc tggggcacca tctggggacc ctgacgtgaa gtttgtcact
     3361 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg
     3421 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg
     3481 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt
     3541 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc
     3601 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt
     3661 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt
     3721 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt
     3781 tagactagta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacgaagct
     3841 tgggcccggg aattaattat taaggccacc atggtgtcta agggcgaaga gctgattaag
     3901 gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc
     3961 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     4021 gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc
     4081 aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag
     4141 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     4201 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     4261 acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg
     4321 ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc
     4381 gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac
     4441 ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac
     4501 aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc
     4561 aaactggggc acaagcttaa tgatctgcag tctagaggat ctggtgccac gaatttctca
     4621 ctactcaagc aggccggtga tgtcgaggaa aaccctggtc ctatgaccga gtacaagccc
     4681 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4741 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4801 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4861 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4921 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4981 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     5041 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     5101 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     5161 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     5221 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgaattcg atatcaagct
     5281 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     5341 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     5401 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     5461 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     5521 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     5581 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     5641 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     5701 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     5761 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5821 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     5881 ataccgtcga cctcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     5941 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     6001 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     6061 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     6121 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     6181 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     6241 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     6301 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     6361 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     6421 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     6481 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     6541 atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct
     6601 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     6661 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     6721 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     6781 agggggagga ttgggaagac aatagcaggc atgtgagcaa aaggccagca aaaggccagg
     6841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6961 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     7021 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     7081 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7141 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7201 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7261 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     7321 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7381 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7441 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7501 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7561 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7621 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7681 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7741 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7801 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7861 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7921 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7981 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     8041 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     8101 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     8161 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     8221 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     8281 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     8341 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     8401 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     8461 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     8521 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     8581 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg a
//
