LOCUS       sequence-200637-        6709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses colicin E1 with R domain deletion and ImmE1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6709)
  AUTHORS   Jakes KS
  TITLE     The Colicin E1 TolC Box: Identification of a Domain Required for 
            Colicin E1 Cytotoxicity and TolC Binding.
  JOURNAL   J Bacteriol. 2016 Dec 13;199(1). pii: e00412-16. Print 2017 Jan 1.
  PUBMED    27795317
REFERENCE   2  (bases 1 to 6709)
  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"; journalName: "J 
            Bacteriol."; date: "2016-12-13"; volume: "199(1). pii"; pages: " 
            e00412-16. Print 2017 Jan 1"
COMMENT     Sequence Label: pKSJ376
FEATURES             Location/Qualifiers
     source          1..6709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1222..1239
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1246..1275
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1757..1775)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1771..1818
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1855..2310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1942..1961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2152..2173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2425..3216,3217..3285))
                     /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     3048..3067
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3286..3378)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3459..4047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3948..3967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4201..4218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4233..4372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4298..4317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4458..4480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4474..4665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5237..6319)
                     /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     6281..6300
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6320..6397)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6603..6622
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gaaaccgcgg tagcgtacta taaagatggt
      121 gttccttatg atgataaggg acaggtaatt attactcttt tgaatggtac tcctgacggg
      181 agtggctctg gcggcggagg tggaaaagga ggcagtaaaa gtgaaagttc tgcagctatt
      241 catgcaactg ctaaatggtc tactgctcaa ttaaagaaaa cacaggcaga gcaggctgcc
      301 cgggcaaaag ctgcagcgga agcacaggcg aaagcaaagg caaacaggga tgcgctgact
      361 cagcgcctga aggatatcgt gaatgaggct cttcgtcaca atgcctcacg tacgccttca
      421 gcaacagagc ttgctcatgc taataatgca gctatgcagg cggaagcaga gcgtttgcgc
      481 cttgcgaaag cagaagaaaa agcccgtaaa gaagcggaag cagcagaaaa ggcttttcag
      541 gaagcagaac aacgacgtaa agagattgaa cgggagaagg ctgaaacaga acgccagttg
      601 aaactggctg aagctgaaga gaaacgactg gctgcattga gtgaagaagc taaagctaat
      661 aatctcctta attcacagat taaggatgct gttgatgcaa cagttagctt ttatcaaacg
      721 ctgactgaaa aatatggtga aaaatattcg aaaatggcac aggaacttgc tgataagtct
      781 aaaggtaaga aaatcggcaa tgtgaatgaa gctctcgctg cttttgaaaa atacaaggat
      841 gttttaaata agaaattcag caaagccgat cgtgatgcta tttttaatgc gttggcatcg
      901 gtgaagtatg atgactgggc taaacattta gatcagtttg ccaagtactt gaagattacg
      961 gggcatgttt cttttggata tgatgtggta tctgatatcc taaaaattaa ggatacaggt
     1021 gactggaagc cactatttct tacattagag aagaaagctg cagatgcagg ggtgagttat
     1081 gttgttgctt tactttttag cttgcttgct ggaactacat taggtatttg gggtattgct
     1141 attgttacag gaattctatg ctcctatatt gataagaata aacttaatac tataaatgag
     1201 gtgttaggga ttgagctcgc tctggtgcca cgcggtagtt ccgctcatca ccaccatcat
     1261 caccatcacc accactaatt aacctagggt gttagggatt taattattct ttattgatat
     1321 aaaaagtcct agcaatccaa atgggattgc taggaccaaa caaagtagat tatatagcat
     1381 aaataggttt aattttgcta cgggggcgtt atttaggttt tttcttcttt cgaaaaaatc
     1441 tttctttatg aagttaaaag ctatgtattc aatagcatat tttgaatatg gacatagaat
     1501 agtgcttatc actattgcat atagcatctt atctgacaca aggaaataat acccttcgct
     1561 gttttttgtt ataaggtata tatatataag tgtgcagtac aggccaaata aaatattttt
     1621 tatgtagtat cttaagctca taaattaaac ctcgccatat attcttttca ttttataagg
     1681 atcgagttat gaggaaaaga ttttttgtgg gaatattcgc gataaacctc cttgttggat
     1741 gcctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1801 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1861 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1921 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1981 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2041 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2101 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2161 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2221 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2281 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     2341 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2401 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2461 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2521 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2581 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2641 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2701 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2761 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2821 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2881 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2941 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3001 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3061 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3121 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3181 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3241 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3301 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3361 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3601 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4081 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4141 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4201 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4261 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4321 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4381 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4441 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4501 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4561 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4621 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4681 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4741 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4801 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4861 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4921 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4981 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5041 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5101 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     5161 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5221 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5341 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5401 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5461 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5521 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5581 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5641 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5701 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5761 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5821 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5881 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5941 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6001 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6061 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6121 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6181 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6241 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6301 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6361 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6421 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6481 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6541 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6601 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6661 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaat
//
