LOCUS       sequence-310528-        5936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses colicin E1 residues 1-190.
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-colE1T
FEATURES             Location/Qualifiers
     source          1..5936
                     /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             60..80
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             664..681
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(734..752)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      748..795
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      832..1287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(919..938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1129..1150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1313..1417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1418..1486,1487..2278)
                     /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(1636..1655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2449..3037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2938..2957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3191..3208
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3223..3362
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3288..3307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3448..3470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3464..3655)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4464..5546)
                     /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     5508..5527
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5547..5624)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5830..5849
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gtaataattt
       61 tgtttaactt taagaaggag gtctagaatg 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 aaatgaggct cttcgtcaca atgcctcacg tacgccttca
      421 gcaacagagc ttgctcatgc taataatgca gctatgcagg cggaagacga gcgtttgcgc
      481 cttgcgaaag cagaagaaaa agcccgtaaa gaagcggaag cagcagaaaa ggcttttcag
      541 gaagcagaac aacgacgtaa agagattgaa cgggagaagg ctgaaacaga acgccagttg
      601 aaactggctg aagctgaaga gaaacgactg gctgcattga gtgaagaagc taaagctctc
      661 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      721 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      781 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      841 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      901 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      961 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1021 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1081 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1141 tccaaactgg aacaacactc aaccatatct cggtctattc ttttgattta taagggattt
     1201 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1261 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1321 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1381 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1441 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1501 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1561 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1621 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1681 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1741 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1801 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1861 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1921 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     1981 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2041 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2101 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     2161 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2221 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2281 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2341 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2401 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2461 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2521 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2581 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2641 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2701 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2761 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2821 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2881 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2941 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3001 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3061 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3121 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3181 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3241 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3301 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3361 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3421 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3481 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3541 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3601 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3661 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3721 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3781 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3841 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3901 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3961 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4021 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4081 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4141 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4201 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4261 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4321 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4381 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4441 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4501 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4561 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4621 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4681 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4741 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4801 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4861 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4921 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4981 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5041 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5101 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5161 tccagcggat agttaataat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5221 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5281 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5341 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5401 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5461 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5521 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5581 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5641 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5701 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5761 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     5821 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5881 gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaat
//
