LOCUS       sequence-337518-        5819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of bdSUMO tagged muGFP with HA2 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-HA2-GFP-HiBit
FEATURES             Location/Qualifiers
     source          1..5819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..32
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        13..31
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             120..142
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             534..1244
                     /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(561..589)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(1361..1379)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1375..1422
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1788..1793,1794..1810,1811..1816))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1843..1861)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1929..2033
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2034..2102,2103..2894)
                     /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(2252..2271)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3065..3653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3554..3573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3807..3824
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3839..3978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3904..3923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4064..4086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4080..4271)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5729..5748
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc tctagtgccg
       61 gctccggaga gctctttaat taagcggccg ccctgcagga ctcgagttct agaaataatt
      121 ttgtttaact ttaagaagga gatatacata tgagcaagca tcaccatcat tcaggccatc
      181 accataccgg acaccaccat cattcaggca gtcatcacca taccggccac atcaatctta
      241 aagtcaaggg acaggacggt aatgaagtgt tctttcgtat caagcgctcg actcagctta
      301 aaaaacttat gaacgcttat tgcgaccgcc aatcggttga tatgacagcc attgcttttt
      361 tatttgatgg ccgtcgtctt cgcgcggaac aaacgcctga tgagcttgag atggaagatg
      421 gggatgagat tgacgccatg ttacaccaga ccggtggggg tctgttcgaa gcgatcgaag
      481 gtttcatcga aaacggttgg gaaggtatga tcgacggttg gtacggtgct agcagcaaag
      541 gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg
      601 ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc tacaaacgga aaactcaccc
      661 ttaaatttat ttgcactact ggaaaactac ctgttccgtg gccaacactt gtcactactc
      721 tgacctatgg tgttctatgc ttttcccgtt atccggatca catgaaacgg catgactttt
      781 tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg
      841 ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg
      901 agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa ctggagtaca
      961 actttaactc acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagctt
     1021 acttcaaaat tcgccacaac gttgaagatg gttccgttca actagcagac cattatcaac
     1081 aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctctcgacac
     1141 aatctgttct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgaggatg
     1201 taactgctgc tgggattaca catggcatgg atgaactcta caaagtatca ggttggcgtc
     1261 ttttcaaaaa gatttccggg ggctcgggct aaggtaccac gcgtgcgcgc tgatccggct
     1321 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1381 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1441 tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag
     1501 tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg
     1561 tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat
     1621 gctgtcggaa tggacgatat cccgcaagag gcccggcagt accagcataa ccaagcctat
     1681 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     1741 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     1801 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     1861 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     1921 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1981 tcatgagaca ataaccctga taaatgcttc aataacattg aaaaaggaag agtatgagta
     2041 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2101 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2161 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2221 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2281 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2341 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2401 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2461 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2521 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2581 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2641 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2701 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2761 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2821 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2881 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2941 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3001 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3061 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3121 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3181 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3241 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3301 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3361 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3421 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3481 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3541 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3601 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3661 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3721 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3781 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3841 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3901 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3961 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4021 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4081 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4141 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4201 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4261 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4321 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4381 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4441 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4501 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4561 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4621 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4681 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
     4741 caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg
     4801 gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct
     4861 ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg
     4921 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc
     4981 ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga
     5041 tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc
     5101 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc
     5161 cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca
     5221 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     5281 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5341 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5401 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5461 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5521 agggcatcgg tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5581 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5641 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5701 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5761 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcg
//
