LOCUS       sequence-358069-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T and R domains of Colicin E1 with N-terminal 40 residue 
            deletion (residues 41-364).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6341)
  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 6341)
  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-TR-del1-40
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1399..1418
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1399..1417
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1418..1442
                     /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             1458..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2467..2484
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2537..2555)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2551..2598
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2635..3090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2722..2741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2932..2953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3116..3220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3221..3289,3290..4081)
                     /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(3439..3458)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4252..4840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4741..4760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4994..5011
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5026..5165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5091..5110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5251..5273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5267..5458)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6267..6341,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaata atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     1381 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1441 cccctctagt aataattttg tttaacttta agaaggaggt ctagaatggg aggcagtaaa
     1501 agtgaaagtt ctgcagctat tcatgcaact gctaaatggt ctactgctca attaaagaaa
     1561 acacaggcag agcaggctgc ccgggcaaaa gctgcagcgg aagcacaggc gaaagcaaag
     1621 gcaaacaggg atgcgctgac tcagcgcctg aaggatatcg taaatgaggc tcttcgtcac
     1681 aatgcctcac gtacgccttc agcaacagag cttgctcatg ctaataatgc agctatgcag
     1741 gcggaagacg agcgtttgcg ccttgcgaaa gcagaagaaa aagcccgtaa agaagcggaa
     1801 gcagcagaaa aggcttttca ggaagcagaa caacgacgta aagagattga acgggagaag
     1861 gctgaaacag aacgccagtt gaaactggct gaagctgaag agaaacgact ggctgcattg
     1921 agtgaagaag ctaaagctgt tgagatcgcc caaaaaaaac tttctgctgc acaatctgaa
     1981 gtggtgaaaa tggatggaga gattaagact ctcaattctc gtttaagctc cagtatccat
     2041 gcccgtgatg cagaaatgaa aacgctcgct ggaaaacgaa atgaactggc tcaggcatcc
     2101 gctaaatata aagaactgga tgagctggtc aaaaaactat caccaagagc caatgatccg
     2161 cttcagaacc gtcctttttt tgaagcaacc agacgacggg ttggggccgg taagattaga
     2221 gaagaaaaac aaaaacaggt aacagcatca gaaacacgta ttaaccggat aaatgctgat
     2281 ataactcaga tccagaaggc tatttctcag gtcagtaata atcgtaatgc cggtatcgct
     2341 cgtgttcatg aagctgaaga aaatttgaaa aaagcacaga ataatctcct taattcacag
     2401 attaaggatg ctgttgatgc aacagttagc ttttatcaaa cgctgactga aaaatatggt
     2461 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     2521 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2581 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     2641 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2701 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2761 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2821 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2881 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2941 tgttccaaac tggaacaaca ctcaaccata tctcggtcta ttcttttgat ttataaggga
     3001 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3061 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3121 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3181 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3241 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3301 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3361 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3421 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3481 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3541 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3601 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3661 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3721 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     3781 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3841 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3901 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3961 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4021 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4081 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4141 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4201 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4261 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4321 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4381 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4441 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4501 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4561 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4621 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4681 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4741 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4801 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4861 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4921 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4981 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     5041 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     5101 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     5161 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5281 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     5341 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     5401 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     5461 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     5521 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     5581 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     5641 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     5701 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     5761 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     5821 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     5881 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     5941 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     6001 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     6061 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     6121 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     6181 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     6241 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6301 gccagctgca ttaatgaatc ggccaacgcg cggggagagg c
//
