LOCUS       sequence-337540-        5819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with pHlip 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-pHlip-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..85
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        66..84
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             173..195
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             587..1297
                     /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(614..642)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1414..1432)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1428..1475
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1841..1846,1847..1863,1864..1869))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1896..1914)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1982..2086
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2087..2155,2156..2947)
                     /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(2305..2324)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3118..3706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3607..3626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3860..3877
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3892..4031
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3957..3976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4117..4139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4133..4324)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5782..5801
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
       61 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg
      121 agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta
      181 actttaagaa ggagatatac atatgagcaa gcatcaccat cattcaggcc atcaccatac
      241 cggacaccac catcattcag gcagtcatca ccataccggc cacatcaatc ttaaagtcaa
      301 gggacaggac ggtaatgaag tgttctttcg tatcaagcgc tcgactcagc ttaaaaaact
      361 tatgaacgct tattgcgacc gccaatcggt tgatatgaca gccattgctt ttttatttga
      421 tggccgtcgt cttcgcgcgg aacaaacgcc tgatgagctt gagatggaag atggggatga
      481 gattgacgcc atgttacacc agaccggtgg ggctgaggag caacaaccat gggctcagta
      541 cctggagttg ttatttccca ctgaaacatt actgttggag tggggcagca aaggagaaga
      601 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
      661 attttctgtc cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt
      721 tatttgcact actggaaaac tacctgttcc gtggccaaca cttgtcacta ctctgaccta
      781 tggtgttcta tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag
      841 tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta
      901 caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa
      961 aggtattgat tttaaagaag atggaaacat tctcggacac aaactggagt acaactttaa
     1021 ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag cttacttcaa
     1081 aattcgccac aacgttgaag atggttccgt tcaactagca gaccattatc aacaaaatac
     1141 tccaattggc gatggccctg tccttttacc agacaaccat tacctctcga cacaatctgt
     1201 tctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagg atgtaactgc
     1261 tgctgggatt acacatggca tggatgaact ctacaaagta tcaggttggc gtcttttcaa
     1321 aaagatttcc gggggctcgg gctaaggtac cacgcgtgcg cgctgatccg gctgctaaca
     1381 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1441 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1501 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     1561 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     1621 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     1681 gaatggacga tatcccgcaa gaggcccggc agtaccagca taaccaagcc tatgcctaca
     1741 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     1801 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     1861 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     1921 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     1981 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2041 acaataaccc tgataaatgc ttcaataaca ttgaaaaagg aagagtatga gtattcaaca
     2101 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2161 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2221 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2281 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     2341 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2401 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2461 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2521 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2581 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     2641 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     2701 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     2761 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     2821 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     2881 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     2941 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3001 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3061 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3121 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3181 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3241 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3301 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3361 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3421 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3481 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3541 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3601 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3661 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3721 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3781 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3841 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3901 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3961 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4021 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4081 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4141 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4201 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4261 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4321 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4381 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4441 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4501 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4561 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4621 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4681 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4741 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     4801 caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg atggatatgt
     4861 tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg gctccaattc
     4921 ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg
     4981 gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgcctacaat
     5041 ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga cgatcagcgg
     5101 tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct gtccctgatg
     5161 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga
     5221 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac
     5281 gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5341 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5401 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5461 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5521 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5581 cggtcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5641 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5701 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5761 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctg
//
