LOCUS       sequence-337533-        5789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with TAT peptide and
            NanoLuc complementation peptide variant #86.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5789)
  AUTHORS   Teo SLY, Rennick JJ, Yuen D, Al-Wassiti H, Johnston APR, Pouton CW
  TITLE     Unravelling cytosolic delivery of cell penetrating peptides with a 
            quantitative endosomal escape assay.
  JOURNAL   Nat Commun. 2021 Jun 17;12(1):3721. doi: 10.1038/s41467-021-23997-x.
  PUBMED    34140497
REFERENCE   2  (bases 1 to 5789)
  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: "2021-06-17"; volume: "12(1):3721. doi"; pages: " 
            10.1038/s41467-021-23997-x"
COMMENT     Sequence Label: bdSUMO-TAT-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     82..101
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     185..204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        185..203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             292..314
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             676..1386
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVLCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAY
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLED
                     VTAAGITHGMDELYK"
     primer_bind     complement(703..731)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1503..1521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1517..1564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1930..1935,1936..1952,1953..1958))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1985..2003)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2071..2175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2176..2244,2245..3036)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2394..2413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3207..3795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3696..3715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3949..3966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3981..4120
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4046..4065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4206..4228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4222..4413)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
       61 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
      121 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
      181 aaattaatac gactcactat agggagacca caacggtttc cctctagtgc cggctccgga
      241 gagctcttta attaagcggc cgccctgcag gactcgagtt ctagaaataa ttttgtttaa
      301 ctttaagaag gagatataca tatgagcaag catcaccatc attcaggcca tcaccatacc
      361 ggacaccacc atcattcagg cagtcatcac cataccggcc acatcaatct taaagtcaag
      421 ggacaggacg gtaatgaagt gttctttcgt atcaagcgct cgactcagct taaaaaactt
      481 atgaacgctt attgcgaccg ccaatcggtt gatatgacag ccattgcttt tttatttgat
      541 ggccgtcgtc ttcgcgcgga acaaacgcct gatgagcttg agatggaaga tggggatgag
      601 attgacgcca tgttacacca gaccggtggg ggtcgtaaaa aacgtcgtca gcgtcgtcgt
      661 ccgccgcagg ctagcagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
      721 gaattagatg gtgatgttaa tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat
      781 gctacaaacg gaaaactcac ccttaaattt atttgcacta ctggaaaact acctgttccg
      841 tggccaacac ttgtcactac tctgacctat ggtgttctat gcttttcccg ttatccggat
      901 cacatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc
      961 actatatctt tcaaagatga cgggacctac aagacgcgtg ctgaagtcaa gtttgaaggt
     1021 gatacccttg ttaatcgtat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     1081 ctcggacaca aactggagta caactttaac tcacacaatg tatacatcac ggcagacaaa
     1141 caaaagaatg gaatcaaagc ttacttcaaa attcgccaca acgttgaaga tggttccgtt
     1201 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     1261 gacaaccatt acctctcgac acaatctgtt ctttcgaaag atcccaacga aaagcgtgac
     1321 cacatggtcc ttcttgagga tgtaactgct gctgggatta cacatggcat ggatgaactc
     1381 tacaaagtat caggttggcg tcttttcaaa aagatttccg ggggctcggg ctaaggtacc
     1441 acgcgtgcgc gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1501 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1561 tttgctgaaa ggaggaacta tatccggata tccacaggac gggtgtggtc gccatgatcg
     1621 cgtagtcgat agtggctcca agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt
     1681 cggacagtgc tccgagaacg ggtgcgcata gaaattgcat caacgcatat agcgctagca
     1741 gcacgccata gtgactggcg atgctgtcgg aatggacgat atcccgcaag aggcccggca
     1801 gtaccagcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     1861 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     1921 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga
     1981 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2041 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2101 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataacat
     2161 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2221 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2281 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2341 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2401 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2461 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2521 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2581 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2641 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2701 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2761 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2821 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2881 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2941 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3001 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3061 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3121 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3181 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3241 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3301 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3361 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3421 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3481 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3541 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3601 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3661 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3721 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3781 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3841 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3901 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3961 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4021 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4081 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4261 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4321 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4381 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4441 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4501 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4561 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4621 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4681 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4741 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4801 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4861 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     4921 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     4981 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     5041 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     5101 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     5161 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     5221 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     5281 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     5341 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     5401 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5461 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5521 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5581 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5641 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
     5701 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5761 gcatgcaagg agatggcgcc caacagtcc
//
