LOCUS       sequence-337538-        5852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with E5TAT peptide 
            and NanoLuc complementation peptide variant #86.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5852)
  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 5852)
  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-E5TAT-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..89
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        70..88
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             177..199
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             624..1334
                     /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(651..679)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1451..1469)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1465..1512
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1878..1883,1884..1900,1901..1906))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1933..1951)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2019..2123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2124..2192,2193..2984)
                     /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(2342..2361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3155..3743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3644..3663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3897..3914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3929..4068
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3994..4013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4154..4176
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4170..4361)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5819..5838
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
       61 ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct agtgccggct
      121 ccggagagct ctttaattaa gcggccgccc tgcaggactc gagttctaga aataattttg
      181 tttaacttta agaaggagat atacatatga gcaagcatca ccatcattca ggccatcacc
      241 ataccggaca ccaccatcat tcaggcagtc atcaccatac cggccacatc aatcttaaag
      301 tcaagggaca ggacggtaat gaagtgttct ttcgtatcaa gcgctcgact cagcttaaaa
      361 aacttatgaa cgcttattgc gaccgccaat cggttgatat gacagccatt gcttttttat
      421 ttgatggccg tcgtcttcgc gcggaacaaa cgcctgatga gcttgagatg gaagatgggg
      481 atgagattga cgccatgtta caccagaccg gtgggggtct gttcgaagcg atcgcggaat
      541 tcatcgaaaa cggttgggaa ggtctgatcg aaggtatggg tcgtaaaaaa cgtcgtcagc
      601 gtcgtcgtcc gccgcaggct agcagcaaag gagaagaact tttcactgga gttgtcccaa
      661 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg
      721 aaggtgatgc tacaaacgga aaactcaccc ttaaatttat ttgcactact ggaaaactac
      781 ctgttccgtg gccaacactt gtcactactc tgacctatgg tgttctatgc ttttcccgtt
      841 atccggatca catgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac
      901 aggaacgcac tatatctttc aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt
      961 ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg tattgatttt aaagaagatg
     1021 gaaacattct cggacacaaa ctggagtaca actttaactc acacaatgta tacatcacgg
     1081 cagacaaaca aaagaatgga atcaaagctt acttcaaaat tcgccacaac gttgaagatg
     1141 gttccgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc
     1201 ttttaccaga caaccattac ctctcgacac aatctgttct ttcgaaagat cccaacgaaa
     1261 agcgtgacca catggtcctt cttgaggatg taactgctgc tgggattaca catggcatgg
     1321 atgaactcta caaagtatca ggttggcgtc ttttcaaaaa gatttccggg ggctcgggct
     1381 aaggtaccac gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga agctgagttg
     1441 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1501 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc
     1561 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc
     1621 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag
     1681 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag
     1741 gcccggcagt accagcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     1801 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     1861 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     1921 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1981 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2041 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2101 aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2161 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2221 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2281 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2341 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2401 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2461 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2521 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2581 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2641 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2701 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2761 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2821 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2881 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2941 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3001 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3061 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3121 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3181 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3241 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3301 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3361 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3421 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3481 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3541 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3601 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3661 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3721 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3781 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3841 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3901 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3961 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4021 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4081 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4141 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4201 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4261 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4321 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4381 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4441 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4501 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4561 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4621 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4681 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4741 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4801 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     4861 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     4921 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     4981 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     5041 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     5101 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     5161 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     5221 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     5281 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     5341 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5401 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5461 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5521 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5581 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct cccttatgcg
     5641 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5701 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5761 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5821 tgatgtcggc gatataggcg ccagcaaccg ca
//
