LOCUS       sequence-337470-        5819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with pHD118 peptide 
            and NanoLuc complementation peptide variant #86.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5819)
  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 5819)
  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-pHD118-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        51..69
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             158..180
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             572..1282
                     /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(599..627)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1399..1417)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1413..1460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1826..1831,1832..1848,1849..1854))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1881..1899)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1967..2071
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2072..2140,2141..2932)
                     /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(2290..2309)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3103..3691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3592..3611
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3845..3862
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3877..4016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3942..3961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4102..4124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4118..4309)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5767..5786
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
       61 cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc tctttaatta
      121 agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt aagaaggaga
      181 tatacatatg agcaagcatc accatcattc aggccatcac cataccggac accaccatca
      241 ttcaggcagt catcaccata ccggccacat caatcttaaa gtcaagggac aggacggtaa
      301 tgaagtgttc tttcgtatca agcgctcgac tcagcttaaa aaacttatga acgcttattg
      361 cgaccgccaa tcggttgata tgacagccat tgctttttta tttgatggcc gtcgtcttcg
      421 cgcggaacaa acgcctgatg agcttgagat ggaagatggg gatgagattg acgccatgtt
      481 acaccagacc ggtgggatcg gcgaagtgct gcatgagttg gccgacgacc tgcctgaatt
      541 acaatcttgg atcaaggctg cgcaacaatt aagcaaagga gaagaacttt tcactggagt
      601 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtccgtgg
      661 agagggtgaa ggtgatgcta caaacggaaa actcaccctt aaatttattt gcactactgg
      721 aaaactacct gttccgtggc caacacttgt cactactctg acctatggtg ttctatgctt
      781 ttcccgttat ccggatcaca tgaaacggca tgactttttc aagagtgcca tgcccgaagg
      841 ttatgtacag gaacgcacta tatctttcaa agatgacggg acctacaaga cgcgtgctga
      901 agtcaagttt gaaggtgata cccttgttaa tcgtatcgag ttaaaaggta ttgattttaa
      961 agaagatgga aacattctcg gacacaaact ggagtacaac tttaactcac acaatgtata
     1021 catcacggca gacaaacaaa agaatggaat caaagcttac ttcaaaattc gccacaacgt
     1081 tgaagatggt tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     1141 ccctgtcctt ttaccagaca accattacct ctcgacacaa tctgttcttt cgaaagatcc
     1201 caacgaaaag cgtgaccaca tggtccttct tgaggatgta actgctgctg ggattacaca
     1261 tggcatggat gaactctaca aagtatcagg ttggcgtctt ttcaaaaaga tttccggggg
     1321 ctcgggctaa ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag
     1381 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1441 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt
     1501 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg
     1561 cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac
     1621 gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc
     1681 cgcaagaggc ccggcagtac cagcataacc aagcctatgc ctacagcatc cagggtgacg
     1741 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     1801 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     1861 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     1921 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1981 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2041 aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2101 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2161 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2221 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2281 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     2341 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2401 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2461 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2521 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2581 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2641 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2701 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     2761 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     2821 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2881 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2941 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3001 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3061 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3121 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3181 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3241 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3301 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3361 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3421 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3481 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3541 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3601 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3661 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3721 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3781 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3841 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3901 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3961 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4021 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4081 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4141 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4201 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4261 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4321 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4381 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4441 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4501 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4561 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4621 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4681 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4741 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     4801 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     4861 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     4921 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     4981 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     5041 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     5101 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     5161 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     5221 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     5281 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5341 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5401 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5461 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5521 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
     5581 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5641 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5701 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5761 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgc
//
