LOCUS       sequence-365364-        5936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T domain of Colicin E1 (residues 1-190) with C terminal 
            cysteine for maleimide chemistry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5936)
  AUTHORS   Budiardjo SJ, Stevens JJ, Calkins AL, Ikujuni AP, Wimalasena VK, 
            Firlar E, Case DA, Biteen JS, Kaelber JT, Slusky JSG
  TITLE     Colicin E1 opens its hinge to plug TolC.
  JOURNAL   Elife. 2022 Feb 24;11. pii: 73297. doi: 10.7554/eLife.73297.
  PUBMED    35199644
REFERENCE   2  (bases 1 to 5936)
  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: "Elife."; 
            date: "2022-02-24"; volume: "11. pii: 73297. doi"; pages: " 
            10.7554/eLife.73297"
COMMENT     Sequence Label: pET303-ColE1-T-E192C
FEATURES             Location/Qualifiers
     source          1..5936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        53..71
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    72..96
                     /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             112..132
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             716..733
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(786..804)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      800..847
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      884..1339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(971..990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1181..1202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1365..1469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1470..1538,1539..2330)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1688..1707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2501..3089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2990..3009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3243..3260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3275..3414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3340..3359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3500..3522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3516..3707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4516..5598)
                     /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     5560..5579
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5599..5676)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5882..5901
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga
       61 ctcactatag gggaattgtg agcggataac aattcccctc tagtaataat tttgtttaac
      121 tttaagaagg aggtctagaa tggaaaccgc ggtagcgtac tataaagatg gtgttcctta
      181 tgatgataag ggacaggtaa ttattactct tttgaatggt actcctgacg ggagtggctc
      241 tggcggcgga ggtggaaaag gaggcagtaa aagtgaaagt tctgcagcta ttcatgcaac
      301 tgctaaatgg tctactgctc aattaaagaa aacacaggca gagcaggctg cccgggcaaa
      361 agctgcagcg gaagcacagg cgaaagcaaa ggcaaacagg gatgcgctga ctcagcgcct
      421 gaaggatatc gtaaatgagg ctcttcgtca caatgcctca cgtacgcctt cagcaacaga
      481 gcttgctcat gctaataatg cagctatgca ggcggaagac gagcgtttgc gccttgcgaa
      541 agcagaagaa aaagcccgta aagaagcgga agcagcagaa aaggcttttc aggaagcaga
      601 acaacgacgt aaagagattg aacgggagaa ggctgaaaca gaacgccagt tgaaactggc
      661 tgaagctgaa gagaaacgac tggctgcatt gagtgaagaa gctaaagctc tctgccacca
      721 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
      781 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
      841 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
      901 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      961 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1021 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1081 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1141 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1201 ggaacaacac tcaaccatat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1261 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1321 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1381 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1441 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1501 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1561 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1621 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1681 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1741 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1801 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1861 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1921 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1981 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     2041 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2101 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2161 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2221 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2281 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2341 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2401 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2461 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2521 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2581 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2641 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2701 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2761 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2821 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2881 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2941 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3001 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3061 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3121 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3181 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3241 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3301 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3361 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3421 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3481 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3541 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3601 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3661 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3721 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3781 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3841 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3901 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3961 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4021 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4081 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4141 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4201 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4261 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4321 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4381 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4441 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4501 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4561 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4621 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4681 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4741 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4801 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4861 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4921 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4981 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5041 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5101 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5161 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5221 atagttaata atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5281 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5341 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5401 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5461 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5521 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5581 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5641 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5701 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5761 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5821 ccatacccac gccgaaacaa gcgctaatga gcccgaagtg gcgagcccga tcttccccat
     5881 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccg
//
