LOCUS       sequence-77957-e        8533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a fibronectin fusion with listeriolysin targeting EGFR in 
            bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8533)
  AUTHORS   Pirie CM, Liu DV, Wittrup KD
  TITLE     Targeted cytolysins synergistically potentiate cytoplasmic delivery 
            of gelonin immunotoxin.
  JOURNAL   Mol Cancer Ther. 2013 Sep;12(9):1774-82. doi: 
            10.1158/1535-7163.MCT-12-1023. Epub 2013 Jul 5.
  PUBMED    23832121
REFERENCE   2  (bases 1 to 8533)
  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: 
            "10.1158/1535-7163.MCT-12-1023"; journalName: "Mol Cancer Ther"; 
            date: "2013-09"; volume: "12"; issue: "9"; pages: "1774-82"
COMMENT     Sequence Label: pMal-E6LLO
FEATURES             Location/Qualifiers
     source          1..8533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /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."
     CDS             81..1163
                     /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     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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)."
     primer_bind     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4624..4641)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4624..4640)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4633..4655)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4819..4836)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4989..5075
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5167..5194
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5213..5304
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5305..5373,5374..6165)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5523..5542)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6207..6720)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6402..6423)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6614..6633
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6831..7419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7320..7339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7573..7590
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7605..7747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7673..7692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7833..7855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7849..8040)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcagaattc
     2701 gctagcgttt ctgatgttcc gagggacctg gaggttgttg ctgcgacccc caccagccta
     2761 ctgatcagct ggttcgacta cgctgtgact tattacagga tcacttacgg agaaacagga
     2821 ggaaatagcc ctgtccagga gttcactgtg cctggttgga tctccactgc taccatcagc
     2881 ggccttaaac ctggagttga ttataccatc actgtgtatg ctgtcactga caactctcgt
     2941 tggccttttc gctctactcc aatttccatt aattaccgaa cagaaattga caaaccatcc
     3001 cagggatccg gaggtggagg tagcaagaaa attatgctgg tttttattac cctgatcctg
     3061 gttagcctgc cgatcgcaca gcagacggaa gcgaaagatg cctctgcatt caacaaagaa
     3121 aacctgatca gctctatggc accgccggcg agcccgccgg cgtctccgaa aaccccgatc
     3181 gagaaaaaac atgccgatga aatcgataaa tacatccagg gcctggatta caacaaaaac
     3241 aacgtgctgg tttaccacgg tgatgcggtg acgaatgttc cgccgcgtaa aggctataaa
     3301 gatggtaacg aatacatcgt ggttgaaaag aaaaagaaaa gcatcaacca gaacaatgcc
     3361 gatattcagg tggttaacgc aatcagtagc ctgacctatc cgggcgcgct ggttaaagcc
     3421 aatagcgaac tggtggaaaa ccagccggat gtgctgccgg ttaaacgtga tagtctgacc
     3481 ctgagcattg atctgccggg tatgacgaac caggataaca aaatcgtggt taaaaacgca
     3541 accaaaagca acgtgaacaa tgcggttaat accctggtgg aacgctggaa cgaaaaatat
     3601 gcgcaggcct acccgaatgt gtctgccaaa attgattatg atgatgaaat ggcctactct
     3661 gaaagtcagc tgatcgcaaa atttggcacc gccttcaaag cagttaacaa tagtctgaac
     3721 gtgaattttg gcgcgattag cgaaggtaaa atgcaggaag aagtgatcag tttcaaacag
     3781 atctactaca acgtgaatgt taacgaaccg acccgtccga gccgcttttt cggcaaagcc
     3841 gttacgaaag aacagctgca ggccctgggt gtgaatgcag aaaacccgcc ggcgtatatt
     3901 tctagtgttg catatggtcg ccaggtgtac ctgaaactgt ctaccaacag ccattctacg
     3961 aaagttaaag cggcctttga tgcagccgtg agtggcaaaa gtgttagcgg tgatgtggaa
     4021 ctgaccaaca tcatcaaaaa cagctctttc aaagcggtta tttatggcgg tagcgccaaa
     4081 gatgaagtgc agattatcga tggcaatctg ggtgatctgc gtgatattct gaaaaaaggc
     4141 gcaaccttta accgcgaaac gccgggtgtt ccgatcgcgt acaccacgaa tttcctgaaa
     4201 gataacgaac tggccgtgat caaaaacaac agcgaataca tcgaaaccac gtctaaagca
     4261 tacaccgatg gcaaaattaa tatcgatcac tctggcggtt atgttgcgca gttcaacatc
     4321 agttgggatg aaatcaacta cgatccggag ggtaacgaaa ttgtgcagca taaaaactgg
     4381 tctgaaaaca acaaaagtaa actggcgcac tttaccagta gcatttatct gccgggcaac
     4441 gcacgtaata tcaacgttta cgcgaaagaa tgcaccggtc tggcctggga atggtggcgt
     4501 acggttattg atgatcgcaa cctgccgctg gtgaaaaatc gcaacattag catctggggc
     4561 accacgctgt atccgaaata ctctaatagt gtggataacc cgatcgaata atagaagctt
     4621 ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4681 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4741 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cagcttggct gttttggcgg
     4801 atgagataag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     4861 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     4921 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     4981 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     5041 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     5101 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     5161 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     5221 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5281 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5341 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5401 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5461 aagatccttg agagttttcg ccccgaagaa cgtttcccaa tgatgagcac ttttaaagtt
     5521 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     5581 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5641 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5701 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5761 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5821 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5881 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5941 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6001 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6061 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6121 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6181 tactcatata tactttagat tgatttaccc cggttgataa tcagaaaagc cccaaaaaca
     6241 ggaagattgt ataagcaaat atttaaattg taaacgttaa tattttgtta aaattcgcgt
     6301 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6361 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6421 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6481 gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6541 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6601 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6661 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6721 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6781 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6841 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6901 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6961 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     7021 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7081 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7141 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7201 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7261 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7321 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7381 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7441 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7501 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7561 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     7621 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     7681 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     7741 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     7801 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     7861 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgcag
     7921 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     7981 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     8041 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     8101 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     8161 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     8221 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     8281 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     8341 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     8401 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     8461 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     8521 gctgcccgaa att
//
