LOCUS       sequence-398274-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 32 Angstrom 4TMD de novo protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  AUTHORS   Peruzzi JA, Gunnels TF, Edelstein HI, Lu P, Baker D, Leonard JN, 
            Kamat NP
  TITLE     Enhancing extracellular vesicle cargo loading and functional 
            delivery by engineering protein-lipid interactions.
  JOURNAL   Nat Commun. 2024 Jul 4;15(1):5618. doi: 10.1038/s41467-024-49678-z.
  PUBMED    38965227
REFERENCE   2  (bases 1 to 7186)
  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: "Nat 
            Commun."; date: "2024-07-4"; volume: "15(1):5618. doi"; pages: " 
            10.1038/s41467-024-49678-z"
COMMENT     Sequence Label: pJPT026-32 Angstrom 4TMD de novo_mRFP1_3x FLAG
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      22..31
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             361..1035
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             361..1035
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(491..509)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     759..778
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     953..976
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1051..1116
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1169..1290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1180..1199)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1234..1253
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1405..1451
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1481..1617
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1662..1733
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1959..2547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2039..2058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3047..4256
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4093..4112
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4260..4859
                     /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(4260..4279)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4756..4776
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4886..4934
                     /note="synthetic polyadenylation signal"
     CDS             join(5200..5268,5269..6060)
                     /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(5418..5437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6073..6209
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6361..6432
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6437..6483)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6501..6549
                     /note="synthetic polyadenylation signal"
     enhancer        6630..6933
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6935..7137
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7088..7108
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gacacacccg ccagcggccg cgccaccatg ggcagcacca gaaccgagat catcacccgg
       61 ctgagcttca gcctgctgct gcaactggtg ctggctatct ttctgctggc cctgctgatc
      121 gtgctgctgt ggctgcttca gcagctgaaa gagctgctga gagagctgga acggctgcag
      181 agagagggaa gctctgacga ggatgtgcgg gaactgctgc gcgagatcaa agaactggtg
      241 gaaaacatcg tgtacctggt tatcatcatc atggtgctgg tgctcgtgat cattgccctg
      301 gccgtgctgc agatgtacct cgtcagggaa ctgaagcggc aggacggatc cggcggctcc
      361 atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc
      421 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      481 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
      541 ctgtcccctc agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc
      601 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
      661 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
      721 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
      781 atgggctggg aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag
      841 atcaagatga ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc
      901 tacatggcca agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac
      961 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc
     1021 cactccaccg gcgcctccgg ctccgaattc gattacaagg accacgatgg cgactataag
     1081 gatcacgaca tcgactacaa ggacgatgac gacaagtgag ctagcttgac tgactgagat
     1141 acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg acaaaccaca
     1201 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1261 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     1321 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     1381 attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa cgggtcttga
     1441 ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc agaaaaaaat
     1501 gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc aacggatacg
     1561 gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta tccttaaggt
     1621 cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt aatggtcttt
     1681 tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc ctaatctcat
     1741 tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt ggcgagatcg
     1801 acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc
     1861 tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa aggccagcaa
     1921 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1981 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2041 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2101 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2161 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2221 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2281 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2341 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2401 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2461 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2521 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2581 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2641 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2701 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2761 ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa
     2821 aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga actatcgcaa
     2881 gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc caggtattac
     2941 tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta catcctcaat
     3001 cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggcc tccgcgccgg
     3061 gttttggcgc ctcccgcggg cgcccccctc gtcacggcga gcgctgccac gtcagacgaa
     3121 gggcgcagga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat
     3181 aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg
     3241 actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc
     3301 tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg
     3361 cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg
     3421 tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt
     3481 ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct
     3541 gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc
     3601 tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag
     3661 gtggggggca tggtgggcgg caagaaccca aggtcttgag cccttcgcta atgcgggaaa
     3721 gctcttattc gggtgagatg ggctgggcac catctgggga ccctgacgtg aagtttgtca
     3781 ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg
     3841 ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc
     3901 tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag gcttttctcc
     3961 gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag gcgccggacc
     4021 tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta tgtacctatc
     4081 ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt
     4141 gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt aattttcagt
     4201 gttagacttg taaattgtcc gctaaattct ggccgttttt ggcttttttg ttagacaaca
     4261 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     4321 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     4381 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     4441 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4501 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4561 cccggctggc cgcgcagcaa cagatggagg gcctcctggc gccgcaccgg cccaaggagc
     4621 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     4681 gcgccgtcgt gctccccgga gtggaggccg ccgagcgcgc cggggtgccc gccttcctgg
     4741 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4801 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctagg
     4861 ctagtatgta agcctagtct tagataataa aatcgctatc catcgaagat ggatgtgtgt
     4921 tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat cctctcagtg
     4981 cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc agcttgggac
     5041 ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg taccgatctg
     5101 tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct agcttttgca
     5161 aacatctatc aagagacagg atcagcagga ggctttcgca tgagtattca acatttccgt
     5221 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5281 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta catcgaactg
     5341 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgctt tccaatgatg
     5401 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5461 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc accagtcaca
     5521 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5581 agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa ggagctaacc
     5641 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5701 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc
     5761 ttgcgtaaac tattaactgg cgaactactt actctagctt cccggcaaca gttgatagac
     5821 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5881 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5941 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6001 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6061 ccgattctag gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac
     6121 tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg
     6181 tgtcctcctt accagaaatt tatccttaag atcccgaatc gtttaaactc gactctggct
     6241 ctatcgaatc tccgtcgttt cgagcttacg cgaacagccg tggcgctcat ttgctcgtcg
     6301 ggcatcgaat ctcgtcagct atcgtcagct tacctttttg gcagcgatcg cggctcccga
     6361 catcttggac cattagctcc acaggtatct tcttccctct agtggtcata acagcagctt
     6421 cagctacctc tcaattcaaa aaacccctca agacccgttt agaggcccca aggggttatg
     6481 ctatcaatcg ttgcgttaca cacacaaaaa accaacacac atccatcttc gatggatagc
     6541 gattttatta tctaactgct gatcgagtgt agccagatct agtaatcaat tacggggtca
     6601 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     6661 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     6721 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     6781 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     6841 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     6901 tacatctacg tattagtcat cgctattacc atgctgatgc ggttttggca gtacatcaat
     6961 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     7021 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     7081 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     7141 ttagtgaacc gtcagatcag atctttgtcg atcctaccat ccactc
//
