LOCUS       sequence-200639-        5683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ImmE1, immunity to colicin E1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5683)
  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 5683)
  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: pKSJ375
FEATURES             Location/Qualifiers
     source          1..5683
                     /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             97..120
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             127..150
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             196..213
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             220..249
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(731..749)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      745..792
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      829..1284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(916..935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1126..1147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1399..2190,2191..2259))
                     /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     2022..2041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2260..2352)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2433..3021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2922..2941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3175..3192
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3207..3346
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3272..3291)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3432..3454
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3448..3639)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4211..5293)
                     /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     5255..5274
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5294..5371)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5577..5596
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gcaagctgga gccacccgca gttcgaaaag
      121 ggtgcacttg aagtcctctt tcagggaccc gggtaccagg atcctgtaca agtcgacgcg
      181 gccgcagagc tcgctctggt gccacgcggt agttccgctc atcaccacca tcatcaccat
      241 caccaccact aattaaccta gggtgttagg gatttaatta ttctttattg atataaaaag
      301 tcctagcaat ccaaatggga ttgctaggac caaacaaagt agattatata gcataaatag
      361 gtttaatttt gctacggggg cgttatttag gttttttctt ctttcgaaaa aatctttctt
      421 tatgaagtta aaagctatgt attcaatagc atattttgaa tatggacata gaatagtgct
      481 tatcactatt gcatatagca tcttatctga cacaaggaaa taataccctt cgctgttttt
      541 tgttataagg tatatatata taagtgtgca gtacaggcca aataaaatat tttttatgta
      601 gtatcttaag ctcataaatt aaacctcgcc atatattctt ttcattttat aaggatcgag
      661 ttatgaggaa aagatttttt gtgggaatat tcgcgataaa cctccttgtt ggatgcctag
      721 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      781 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      841 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      901 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      961 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1021 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1081 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1141 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1201 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1261 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     1321 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     2041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     2281 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2341 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2401 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2461 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2521 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2581 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2641 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2701 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2761 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2821 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2881 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2941 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3001 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3061 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3121 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3181 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3241 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3301 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3361 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3421 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3481 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3541 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3601 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3661 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3721 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3781 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3841 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3901 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     3961 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4021 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4081 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4141 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4201 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4261 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4321 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4381 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4441 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     4501 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4561 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4621 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4681 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4741 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4801 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4861 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4921 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4981 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5041 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5101 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5161 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5221 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5281 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5341 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5401 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5461 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5521 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5581 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5641 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aat
//
