LOCUS       sequence-481076-        6758 bp    DNA     circular SYN 29-JUL-2025
DEFINITION  Expresses micelle-forming elastin-like polypeptide (ELP)-endosomal 
            escape peptide (EEP) fusion construct in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6758)
  AUTHORS   Eweje F, Ibrahim V, Shajii A, Walsh ML, Ahmad K, Alrefai A, Miyasato
            D, Davis JR, Ham H, Li K, Roehrl M, Haller CA, Liu DR, Chen J, 
            Chaikof EL
  TITLE     Self-assembling protein nanoparticles for cytosolic delivery of 
            nucleic acids and proteins.
  JOURNAL   Nat Biotechnol. 2025 May 15. doi: 10.1038/s41587-025-02664-2.
  PUBMED    40374955
REFERENCE   2  (bases 1 to 6758)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 May 15. doi: 10.1038/s41587-025-02664-2."
COMMENT     Sequence Label: pQE-80L-V4-ELP-EEP13
FEATURES             Location/Qualifiers
     source          1..6758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    62..78
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             136..153
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2215..2309
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2353..3012
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2419..2438)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3077..3163
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3256..4338)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4300..4319
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4339..4416)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4500..4522)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4608..4748
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4663..4682
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4763..4780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4934..5522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5014..5033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5693..6484,6485..6553))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6316..6335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6554..6658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6726..6744
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgggc
      121 ggtggccgtg gttctcatca ccatcaccat cacggatccg tacccggggc tggtgttcca
      181 ggaggcggag tcccgggtgc cggcgtgcca ggtggtggcg ttccgggtgc gggtgttcct
      241 ggcgcgggtg ttccgggtgg tggtgtgcca ggcgcgggtg tgccgggtgg tggcgttccg
      301 ggtgccggtg tgccgggtgc cggtgtaccg ggtggtggcg tcccaggcgc gggtgttccg
      361 ggcggtggcg tcccgggtgc tggcgttccg ggtgcaggcg ttccgggcgg tggtgtcccg
      421 ggtgcaggcg tgccgggcgg gggcgtgccg ggtgctggcg tgccgggtgc tggcgtgcct
      481 ggcggaggcg tcccgggcgc tggcgtgccg ggtggtggcg tgccgggtgc cggtgttccg
      541 ggagcagggg tccctggtgg cggcgttccg ggtgcgggcg ttccgggcgg aggcgttccg
      601 ggcgcgggtg ttccgggtgc aggcgtgcca ggtggtggcg tgccgggcgc gggtgttccg
      661 ggtggcggcg ttccgggcgc gggtgtgccg ggtgcaggcg ttccgggtgg cggggttccg
      721 ggtgcgggtg ttccgggcgg aggcgtgccg ggtgcgggtg tgccgggcgc gggtgtaccg
      781 ggcggcggcg ttccgggcgc tggcgtgccg ggtggtggcg tcccaggcgc aggcgtaccc
      841 ggggctggtg ttccaggagg cggagtcccg ggtgccggcg tgccaggtgg tggggtaccg
      901 ggtgccggct gttgctgttg cggtggcggt ataccaggtc atggcatccc gggtcacggt
      961 gtaccaggct atggtattcc gggtcacggc atcccaggcc atggtattcc aggtcatggc
     1021 atcccgggtc acggtgtacc aggctatggt attccgggtc acggcatccc aggccatggt
     1081 attccaggtc atggcatccc gggtcacggt gtaccaggct atggtattcc gggtcacggc
     1141 atcccaggcc atggtattcc aggtcatggc atcccgggtc acggtgtacc aggctatggt
     1201 attccgggtc acggcatccc aggccatggt attccaggtc atggcatccc gggtcacggt
     1261 gtaccaggct atggtattcc gggtcacggc atcccaggcc atggtattcc aggtcatggc
     1321 atcccgggtc acggtgtacc aggctatggt attccgggtc acggcatccc aggccatggt
     1381 attccaggtc atggcatccc gggtcacggt gtaccaggct atggtattcc gggtcacggc
     1441 atcccaggcc atggtattcc aggtcatggc atcccgggtc acggtgtacc aggctatggt
     1501 attccgggtc acggcatccc aggccatggt attccaggtc atggcatccc gggtcacggt
     1561 gtaccaggct atggtattcc gggtcacggc atcccaggcc atggtattcc aggtcatggc
     1621 atcccgggtc acggtgtacc aggctatggt attccgggtc acggcatccc aggccatggt
     1681 attccaggtc atggcatccc gggtcacggt gtaccaggct atggtattcc gggtcacggc
     1741 atcccaggcc atggtattcc aggtcatggc atcccgggtc acggtgtacc aggctatggt
     1801 attccgggtc acggcatccc aggccatggt attccaggtc atggcatccc gggtcacggt
     1861 gtaccaggct atggtattcc gggtcacggc atcccaggcc atggtattcc aggtcatggc
     1921 atcccgggtc acggtgtacc aggctatggt attccgggtc acggcatccc aggccatggt
     1981 attccaggtc atggcatccc gggtcacggt gtaccaggct atggtattcc gggtcacggc
     2041 atcccaggcc atggtattcc aggctatggt aagaattgga aaggaatagc tgggatggca
     2101 aagaagttgc tgggcaagaa ctggggtggt agcggtggcg gctccgtgaa ttggaaaaag
     2161 atcctgggta aaatcattaa agttgtcaaa taaaagctta attagctgag cttggactcc
     2221 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
     2281 gccgccgggc gttttttatt ggtgagaatc caagctagct tggcgagatt ttcaggagct
     2341 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     2401 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     2461 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     2521 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca
     2581 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     2641 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     2701 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     2761 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     2821 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     2881 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     2941 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     3001 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact
     3061 ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     3121 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagattacg
     3181 tgcagtcgat gataagctgt caaacatgag aattgtgcct aatgagtgag ctaacttaca
     3241 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3301 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3361 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3421 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3481 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     3541 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3601 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     3661 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     3721 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     3781 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     3841 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     3901 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     3961 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4021 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4081 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4141 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4201 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4261 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4321 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4381 cataccgcga aaggttttgc accattcgat ggtgtcggaa tttcgggcag cgttgggtcc
     4441 tggccacggg tgcgcatgat ctagagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     4501 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     4561 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     4621 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     4681 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     4741 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5341 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6661 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6721 ataaaaatag gcgtatcacg aggccctttc gtcttcac
//
