LOCUS       sequence-477208-        8816 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 8816)
  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 8816)
  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-NeoR
FEATURES             Location/Qualifiers
     source          1..8816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2622..3833
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3670..3689
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3864..3873
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3870..4580
                     /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             4605..4661
                     /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             4656..5456
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="GPMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4716..4735)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5326..5345
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    5482..6070
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5535..5555)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5953..5964)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6073..6089)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6074..6090)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6595..6775
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6801..6818)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6807..6918
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(7064..7652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7144..7163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7823..8614,8615..8683))
                     /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     8446..8465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8684..8788)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta acccgtgtcg gctccagatc tggcctccgc gccgggtttt
     2641 ggcgcctccc gcgggcgccc ccctcctcac ggcgagcgct gccacgtcag acgaagggcg
     2701 cagcgagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga
     2761 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc
     2821 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg
     2881 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc
     2941 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga
     3001 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc
     3061 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt
     3121 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt
     3181 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg
     3241 ggggcatggt gggcggcaag aacccaaggt cttgaggcct tcgctaatgc gggaaagctc
     3301 ttattcgggt gagatgggct ggggcaccat ctggggaccc tgacgtgaag tttgtcactg
     3361 actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc
     3421 agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt
     3481 tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc
     3541 gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct
     3601 ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc
     3661 ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg
     3721 aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt
     3781 agactagtaa attgtccgct aaattctggc cgtttttggc ttttttgtta gacgaagctt
     3841 gggcccggga attaattatt aaggccacca tggtgtctaa gggcgaagag ctgattaagg
     3901 agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca
     3961 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     4021 agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca
     4081 agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg
     4141 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     4201 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca
     4261 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc
     4321 tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg
     4381 ggagccatct gatcgcaaac gccaagacca catatagatc caagaaaccc gctaagaacc
     4441 tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca
     4501 acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca
     4561 aactggggca caagcttaat gatctgcagt ctagaggatc tggtgccacg aatttctcac
     4621 tactcaagca ggccggtgat gtcgaggaaa accctggtcc tatgattgaa caagatggat
     4681 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4741 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4801 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4861 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4921 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4981 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     5041 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     5101 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     5161 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat ctcgtcgtga
     5221 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     5281 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     5341 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     5401 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgacgaa
     5461 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     5521 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5581 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5641 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     5701 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5761 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5821 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     5881 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5941 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     6001 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     6061 gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt
     6121 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
     6181 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
     6241 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     6301 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
     6361 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
     6421 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
     6481 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
     6541 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
     6601 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     6661 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     6721 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     6781 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6841 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6901 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6961 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgtga gcaaaaggcc
     7021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     7081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     7141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7741 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7801 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7861 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7921 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7981 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     8041 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     8101 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     8161 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     8221 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     8281 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     8341 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     8401 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     8461 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8521 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8581 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8641 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8701 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8761 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgac
//
