LOCUS       sequence-337537-        5858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with 5.3 peptide and
            NanoLuc complementation peptide variant #86.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  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 5858)
  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-5.3-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5858
                     /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             634..1344
                     /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(661..689)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1461..1479)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1475..1522
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1888..1893,1894..1910,1911..1916))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1943..1961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2029..2133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2134..2202,2203..2994)
                     /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(2352..2371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3165..3753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3654..3673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3907..3924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3939..4078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4004..4023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4164..4186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4180..4371)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5829..5848
                     /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 accggtgggg gtccgtctca gccgacctac
      541 cctggtgacg acgcgccggt tcgtgacctg atccgtttct accgtgacct gcgtcgttac
      601 ctgaacgttg ttacccgtca ccgttacgct agcagcaaag gagaagaact tttcactgga
      661 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtccgt
      721 ggagagggtg aaggtgatgc tacaaacgga aaactcaccc ttaaatttat ttgcactact
      781 ggaaaactac ctgttccgtg gccaacactt gtcactactc tgacctatgg tgttctatgc
      841 ttttcccgtt atccggatca catgaaacgg catgactttt tcaagagtgc catgcccgaa
      901 ggttatgtac aggaacgcac tatatctttc aaagatgacg ggacctacaa gacgcgtgct
      961 gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg tattgatttt
     1021 aaagaagatg gaaacattct cggacacaaa ctggagtaca actttaactc acacaatgta
     1081 tacatcacgg cagacaaaca aaagaatgga atcaaagctt acttcaaaat tcgccacaac
     1141 gttgaagatg gttccgttca actagcagac cattatcaac aaaatactcc aattggcgat
     1201 ggccctgtcc ttttaccaga caaccattac ctctcgacac aatctgttct ttcgaaagat
     1261 cccaacgaaa agcgtgacca catggtcctt cttgaggatg taactgctgc tgggattaca
     1321 catggcatgg atgaactcta caaagtatca ggttggcgtc ttttcaaaaa gatttccggg
     1381 ggctcgggct aaggtaccac gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga
     1441 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1501 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg
     1561 gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg
     1621 ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca
     1681 acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat
     1741 cccgcaagag gcccggcagt accagcataa ccaagcctat gcctacagca tccagggtga
     1801 cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt
     1861 agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat
     1921 gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1981 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2101 taaatgcttc aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2341 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     2401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2641 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2821 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3301 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3781 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3841 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3901 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3961 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4021 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4081 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4141 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4201 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4261 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4321 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4381 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4441 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4501 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4561 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4621 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4681 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4741 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4801 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
     4861 gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt
     4921 ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa
     4981 tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg
     5041 cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg
     5101 ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa
     5161 gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc
     5221 tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc
     5281 ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg
     5341 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     5401 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     5461 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     5521 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     5581 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct
     5641 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5701 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5761 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5821 tccccatcgg tgatgtcggc gatataggcg ccagcaac
//
