LOCUS       sequence-373984-        6908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Internalization and Endosomal Escape to effectively route a protein 
            cargo to the cytosol of cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  AUTHORS   Mohammed AF, Abdul-Wahid A, Huang EH, Bolewska-Pedyczak E, Cydzik M,
            Broad AE, Gariepy J
  TITLE     The Pseudomonas aeruginosa exotoxin A translocation domain 
            facilitates the routing of CPP-protein cargos to the cytosol of 
            eukaryotic cells.
  JOURNAL   J Control Release. 2012 Nov 28;164(1):58-64. doi: 
            10.1016/j.jconrel.2012.10.006. Epub 2012 Oct 14.
  PUBMED    23075769
REFERENCE   2  (bases 1 to 6908)
  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"; doi: 
            "10.1016/j.jconrel.2012.10.006"; journalName: "J Control Release"; 
            date: "2012-11-28- 28"; volume: "164"; issue: "1"; pages: "58-64"
COMMENT     Sequence Label: Pet15b-Internalization and Endosomal Escape
FEATURES             Location/Qualifiers
     source          1..6908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(186..1268)
                     /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     1230..1249
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1269..1346)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1552..1571
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1655..1674
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1655..1673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1674..1698
                     /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             1713..1735
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1778..1801
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(2282..2284,2285..2287,2288..3004)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKD"
     primer_bind     complement(2327..2348)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2588..2607)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2935..2956
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3063..3081)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3077..3124
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3299..3304,3305..3321,3322..3327))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3354..3372)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3440..3544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3545..3613,3614..4405)
                     /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(3763..3782)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4576..5164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5065..5084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5318..5335
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5350..5489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5415..5434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5575..5597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5591..5782)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
       61 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
      121 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
      181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
      301 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
      361 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
      421 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
      481 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
      541 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
      601 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
      661 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
      721 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
      781 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
      841 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      901 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      961 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     1021 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     1081 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     1141 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     1201 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     1261 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     1321 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     1381 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     1441 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     1501 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     1561 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     1621 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1681 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1741 catgggccgc cgtcgccgcc gtcgtcgccg ccgccgtcat catcatcatc atcatcatca
     1801 cggcggcagc ctggccgcgc tgaccgcgca ccaggcttgc cacctgccgc tggagacttt
     1861 cacccgtcat cgccagccgc gcggctggga acaactggag cagtgcggct atccggtgca
     1921 gcggctggtc gccctctacc tggcggcgcg gctgtcgtgg aaccaggtcg accaggtgat
     1981 ccgcaacgcc ctggccagcc ccggcagcgg cggcgacctg ggcgaagcga tccgcgagca
     2041 gccggagcag gcccgtctgg ccctgaccct ggccgccgcc gagagcgagc gcttcgtccg
     2101 gcagggcacc ggcaacgacg aggccggcgc ggccaacgcc gacgtggtga gcctgacctg
     2161 cccggtcgcc gccggtgaat gcgcgggccc ggcggacagc ggcgacgccc tgctggagcg
     2221 caactatccc actggcgcgg agttcctcgg cgacggcggc gacgtcagct tcagcacccg
     2281 tatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2341 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2401 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2461 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2521 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2581 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2641 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2701 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2761 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2821 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2881 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2941 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     3001 ttgatgactc gaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3061 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3121 tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata
     3181 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     3241 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     3301 taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt
     3361 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3421 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3481 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3541 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3601 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3661 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3721 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3781 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3841 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3901 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3961 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4021 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4081 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4141 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4201 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4261 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4321 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4381 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4441 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4501 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4561 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4621 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4681 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     4741 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4801 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4861 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4921 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4981 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5041 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5101 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5161 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5221 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5281 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5341 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     5401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     5461 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     5521 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5581 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     5641 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     5701 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     5761 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     5821 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     5881 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     5941 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     6001 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     6061 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     6121 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     6181 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     6241 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg
     6301 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga
     6361 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat
     6421 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta
     6481 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat
     6541 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc
     6601 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg
     6661 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt
     6721 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg
     6781 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     6841 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     6901 gccgaaaa
//
