LOCUS       sequence-373988-        6428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative Control mediating endosomal localization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6428)
  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 6428)
  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 Control
FEATURES             Location/Qualifiers
     source          1..6428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(63..1145)
                     /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     1107..1126
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1146..1223)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1429..1448
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1532..1551
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1532..1550
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1551..1575
                     /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             1590..1612
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1655..1678
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(1679..1681,1682..1684,1685..2401)
                     /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(1724..1745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1985..2004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2332..2353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2460..2478)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2474..2521
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2696..2701,2702..2718,2719..2724))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2751..2769)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2837..2941
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2942..3010,3011..3802)
                     /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(3160..3179)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3973..4561
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4462..4481
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4715..4732
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4747..4886
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4812..4831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4972..4994
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4988..5179)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
       61 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
      121 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
      181 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
      241 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
      301 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
      361 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
      421 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
      481 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
      541 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
      601 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
      661 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
      721 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
      781 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
      841 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
      901 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
      961 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     1021 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     1081 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     1141 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     1201 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     1261 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     1321 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     1381 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     1441 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     1501 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     1561 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat
     1621 gggccgccgt cgccgccgtc gtcgccgccg ccgtcatcat catcatcatc atcatcacat
     1681 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1741 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1801 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1861 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1921 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1981 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2041 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2101 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2161 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2221 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2281 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2341 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggattg
     2401 atgactcgag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2461 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2521 gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc
     2581 aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta
     2641 gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa
     2701 agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat
     2761 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2821 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2881 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2941 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3001 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3061 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3121 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3181 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3241 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3301 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3361 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3421 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3481 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3541 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3601 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3661 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3721 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3781 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3841 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3901 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3961 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4021 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4081 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4141 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4201 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4261 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4321 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4381 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4441 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4501 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4561 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4621 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4681 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4741 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4801 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4861 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4921 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4981 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5041 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5101 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5161 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5221 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5281 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5341 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5401 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5461 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5521 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5581 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5641 cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg
     5701 acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt
     5761 gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca
     5821 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag
     5881 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc
     5941 cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg
     6001 aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat
     6061 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc
     6121 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
     6181 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     6241 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     6301 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     6361 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     6421 ggctctca
//
