LOCUS       sequence-310524-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses signal sequence-less TolC as inclusion bodies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  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 5648)
  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: pTrc99a-NoSS-TolC
FEATURES             Location/Qualifiers
     source          1..5648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..30)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    38..54
                     /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     43..65
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(1640..1657)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1810..1896
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1988..2015
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2034..2125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2126..2194,2195..2986)
                     /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(2344..2363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3157..3745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3646..3665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3899..3916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3931..4071
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3997..4016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4157..4179
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4257..4334
                     /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             4335..5417
                     /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(4354..4373)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac
       61 acaggaaaca gaccatgagg aaattgctcc ccattcttat cggcctgagc ctttctgggt
      121 tcagttcgtt gagccaggcc gagaacctga tgcaagttta tcagcaagca cgccttagta
      181 acccggaatt gcgtaagtct gccgccgatc gtgatgctgc ctttgaaaaa attaatgaag
      241 cgcgcagtcc attactgcca cagctaggtt taggtgcaga ttacacctat agcaacggct
      301 accgcgacgc gaacggcatc aactctaacg cgaccagtgc gtccttgcag ttaactcaat
      361 ccatttttga tatgtcgaaa tggcgtgcgt taacgctgca ggaaaaagca gcagggattc
      421 aggacgtcac gtatcagacc gatcagcaaa ccttgatcct caacaccgcg accgcttatt
      481 tcaacgtgtt gaatgctatt gacgttcttt cctatacaca ggcacaaaaa gaagcgatct
      541 accgtcaatt agatcaaacc acccaacgtt ttaacgtggg cctggtagcg atcaccgacg
      601 tgcagaacgc ccgcgcacag tacgataccg tgctggcgaa cgaagtgacc gcacgtaata
      661 accttgataa cgcggtagag cagctgcgcc agatcaccgg taactactat ccggaactgg
      721 ctgcgctgaa tgtcgaaaac tttaaaaccg acaaaccaca gccggttaac gcgctgctga
      781 aagaagccga aaaacgcaac ctgtcgctgt tacaggcacg cttgagccag gacctggcgc
      841 gcgagcaaat tcgccaggcg caggatggtc acttaccgac tctggattta acggcttcta
      901 ccgggatttc tgacacctct tatagcggtt cgaaaacccg tggtgccgct ggtacccagt
      961 atgacgatag caatatgggc cagaacaaag ttggcctgag cttctcgctg ccgatttatc
     1021 agggcggaat ggttaactcg caggtgaaac aggcacagta caactttgtc ggtgccagcg
     1081 agcaactgga aagtgcccat cgtagcgtcg tgcagaccgt gcgttcctcc ttcaacaaca
     1141 ttaatgcatc tatcagtagc attaacgcct acaaacaagc cgtagtttcc gctcaaagct
     1201 cattagacgc gatggaagcg ggctactcgg tcggtacgcg taccattgtt gatgtgttgg
     1261 atgcgaccac cacgttgtac aacgccaagc aagagctggc gaatgcgcgt tataactacc
     1321 tgattaatca gctgaatatt aagtcagctc tgggtacgtt gaacgagcag gatctgctgg
     1381 cactgaacaa tgcgctgagc aaaccggttt ccactaatcc ggaaaacgtt gcaccgcaaa
     1441 cgccggaaca gaatgctatt gctgatggtt atgcgcctga tagcccggca ccagtcgttc
     1501 agcaaacatc cgcacgcact accaccagta acggtcataa ccctttccgt aactgatgac
     1561 gacgacgggg cttcggcccc gtctgaacgt aaggcaacgt aaagcttggc tgttttggcg
     1621 gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa
     1681 aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag
     1741 aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact
     1801 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     1861 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     1921 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
     1981 attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt
     2041 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2101 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2161 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2221 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2281 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2341 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     2401 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2461 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2521 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2581 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2641 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     2701 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2761 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2821 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2881 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2941 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3001 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3061 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3121 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3181 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3241 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3301 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3361 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3421 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3481 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3541 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3601 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3661 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3721 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3781 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3841 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3901 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3961 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     4021 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4081 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4141 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4201 cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca
     4261 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     4321 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     4381 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4441 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4501 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4561 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4621 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4681 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4741 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4801 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4861 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4921 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4981 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     5041 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     5101 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     5161 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     5221 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     5281 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     5341 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5401 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt gatctggttt
     5461 gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     5521 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     5581 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
     5641 atgagctg
//
