LOCUS       sequence-373987-        6911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative Control confirming the need for a CPP peptide in mediating 
            internalization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6911)
  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 6911)
  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-Endosomal Escape Control
FEATURES             Location/Qualifiers
     source          1..6911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(746..1828)
                     /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     1790..1809
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1829..1906)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2112..2131
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2215..2234
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2215..2233
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2234..2258
                     /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             2273..2295
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2314..2331
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2341..2358
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(2845..2847,2848..2850,2851..3567)
                     /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(2890..2911)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3151..3170)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3498..3519
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3626..3644)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3640..3687
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3862..3867,3868..3884,3885..3890))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3917..3935)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4003..4107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4108..4176,4177..4968)
                     /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(4326..4345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5139..5727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5628..5647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5881..5898
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5913..6052
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5978..5997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6138..6160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6154..6345)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
       61 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
      121 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
      181 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
      241 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
      301 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
      361 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
      421 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
      481 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
      541 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
      601 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
      661 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
      721 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      781 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
      841 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
      901 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      961 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     1021 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     1081 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     1141 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     1201 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     1261 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     1321 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     1381 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     1441 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     1501 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     1561 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     1621 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     1681 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     1741 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     1801 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1861 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1921 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1981 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     2041 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     2101 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     2161 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     2221 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     2281 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
     2341 ctggtgccgc gcggcagcca tatgggcggc agcctggccg cgctgaccgc gcaccaggct
     2401 tgccacctgc cgctggagac tttcacccgt catcgccagc cgcgcggctg ggaacaactg
     2461 gagcagtgcg gctatccggt gcagcggctg gtcgccctct acctggcggc gcggctgtcg
     2521 tggaaccagg tcgaccaggt gatccgcaac gccctggcca gccccggcag cggcggcgac
     2581 ctgggcgaag cgatccgcga gcagccggag caggcccgtc tggccctgac cctggccgcc
     2641 gccgagagcg agcgcttcgt ccggcagggc accggcaacg acgaggccgg cgcggccaac
     2701 gccgacgtgg tgagcctgac ctgcccggtc gccgccggtg aatgcgcggg cccggcggac
     2761 agcggcgacg ccctgctgga gcgcaactat cccactggcg cggagttcct cggcgacggc
     2821 ggcgacgtca gcttcagcac ccgtatggtg agcaagggcg aggagctgtt caccggggtg
     2881 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2941 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3001 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     3061 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3121 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3181 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3241 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3301 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3361 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3421 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3481 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3541 ggcatggacg agctgtacaa ggattgatga ctcgaggatc cggctgctaa caaagcccga
     3601 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3661 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca
     3721 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     3781 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     3841 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     3901 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3961 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4021 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4081 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4141 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4201 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4261 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4321 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     4381 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4441 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4501 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4561 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4621 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4681 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4741 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4801 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4861 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4921 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4981 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5041 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5101 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5161 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5221 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5281 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5341 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5401 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5461 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5521 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5581 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5641 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5701 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5761 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5821 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5881 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5941 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     6001 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     6061 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6121 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6181 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6241 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6301 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     6361 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6421 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     6481 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6541 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6601 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6661 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6721 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6781 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     6841 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     6901 cgcattcaca g
//
