LOCUS       sequence-337536-        5834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with ZF5.3 peptide 
            and NanoLuc complementation peptide variant #86.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5834)
  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 5834)
  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-ZF5.3-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        74..92
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             181..203
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             610..1320
                     /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(637..665)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1437..1455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1451..1498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1864..1869,1870..1886,1887..1892))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1919..1937)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2005..2109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2110..2178,2179..2970)
                     /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(2328..2347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3141..3729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3630..3649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3883..3900
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3915..4054
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3980..3999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4140..4162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4156..4347)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5805..5824
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
       61 gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagtgcc
      121 ggctccggag agctctttaa ttaagcggcc gccctgcagg actcgagttc tagaaataat
      181 tttgtttaac tttaagaagg agatatacat atgagcaagc atcaccatca ttcaggccat
      241 caccataccg gacaccacca tcattcaggc agtcatcacc ataccggcca catcaatctt
      301 aaagtcaagg gacaggacgg taatgaagtg ttctttcgta tcaagcgctc gactcagctt
      361 aaaaaactta tgaacgctta ttgcgaccgc caatcggttg atatgacagc cattgctttt
      421 ttatttgatg gccgtcgtct tcgcgcggaa caaacgcctg atgagcttga gatggaagat
      481 ggggatgaga ttgacgccat gttacaccag accggtgggt ggtactcttg caacgtttgc
      541 ggtaaagcgt tcgttctgtc tcgtcacctg aaccgtcacc tgcgtgttca ccgtcgtgcg
      601 accgctagca gcaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
      661 gatggtgatg ttaatgggca caaattttct gtccgtggag agggtgaagg tgatgctaca
      721 aacggaaaac tcacccttaa atttatttgc actactggaa aactacctgt tccgtggcca
      781 acacttgtca ctactctgac ctatggtgtt ctatgctttt cccgttatcc ggatcacatg
      841 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga acgcactata
      901 tctttcaaag atgacgggac ctacaagacg cgtgctgaag tcaagtttga aggtgatacc
      961 cttgttaatc gtatcgagtt aaaaggtatt gattttaaag aagatggaaa cattctcgga
     1021 cacaaactgg agtacaactt taactcacac aatgtataca tcacggcaga caaacaaaag
     1081 aatggaatca aagcttactt caaaattcgc cacaacgttg aagatggttc cgttcaacta
     1141 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     1201 cattacctct cgacacaatc tgttctttcg aaagatccca acgaaaagcg tgaccacatg
     1261 gtccttcttg aggatgtaac tgctgctggg attacacatg gcatggatga actctacaaa
     1321 gtatcaggtt ggcgtctttt caaaaagatt tccgggggct cgggctaagg taccacgcgt
     1381 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1441 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1501 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     1561 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     1621 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     1681 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtacca
     1741 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     1801 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     1861 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     1921 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1981 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2041 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata acattgaaaa
     2101 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2161 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2221 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2281 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2341 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2401 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2461 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2521 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2581 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2641 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2701 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2761 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2821 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2881 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2941 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3001 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3061 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3121 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3181 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3241 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3301 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3361 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3421 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3481 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3541 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3601 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3661 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3721 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3781 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3841 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3901 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3961 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4021 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4081 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4141 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4201 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4261 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4321 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4381 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4441 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4501 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4561 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4621 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4681 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4741 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4801 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     4861 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     4921 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     4981 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     5041 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     5101 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     5161 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     5221 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     5281 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     5341 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5401 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5461 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5521 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5581 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     5641 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5701 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5761 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5821 taggcgccag caac
//
