LOCUS       sequence-310529-        6458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses colicin E1 residues 1-364.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6458)
  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 6458)
  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-colE1TR
FEATURES             Location/Qualifiers
     source          1..6458
                     /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             1186..1203
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1256..1274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1270..1317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1354..1809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1441..1460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1651..1672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1835..1939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1940..2008,2009..2800)
                     /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(2158..2177)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2971..3559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3460..3479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3713..3730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3745..3884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3810..3829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3970..3992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3986..4177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4986..6068)
                     /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     6030..6049
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6069..6146)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6352..6371
                     /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 taaagctgtt
      661 gagatcgccc aaaaaaaact ttctgctgca caatctgaag tggtgaaaat ggatggagag
      721 attaagactc tcaattctcg tttaagctcc agtatccatg cccgtgatgc agaaatgaaa
      781 acgctcgctg gaaaacgaaa tgaactggct caggcatccg ctaaatataa agaactggat
      841 gagctggtca aaaaactatc accaagagcc aatgatccgc ttcagaaccg tccttttttt
      901 gaagcaacca gacgacgggt tggggccggt aagattagag aagaaaaaca aaaacaggta
      961 acagcatcag aaacacgtat taaccggata aatgctgata taactcagat ccagaaggct
     1021 atttctcagg tcagtaataa tcgtaatgcc ggtatcgctc gtgttcatga agctgaagaa
     1081 aatttgaaaa aagcacagaa taatctcctt aattcacaga ttaaggatgc tgttgatgca
     1141 acagttagct tttatcaaac gctgactgaa aaatatggtc tcgagcacca ccaccaccac
     1201 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1261 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1321 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1381 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1441 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1501 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1561 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1621 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1681 tcaaccatat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1741 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1801 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1861 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1921 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1981 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2041 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2101 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2161 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     2221 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2281 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2341 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2401 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     2461 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2521 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2581 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2641 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2701 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2761 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2821 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2881 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2941 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3001 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3061 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3121 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3181 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3241 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3301 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3361 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3421 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3481 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3541 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3601 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3661 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3721 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3781 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3841 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3901 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3961 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4021 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4081 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4141 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4201 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4261 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4321 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4381 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4441 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4501 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4561 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4621 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     4681 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4741 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4801 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4861 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4921 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4981 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5041 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     5101 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     5161 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     5221 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     5281 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     5341 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     5401 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5461 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     5521 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     5581 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     5641 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaata
     5701 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     5761 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5821 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5881 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5941 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     6001 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     6061 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     6121 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     6181 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     6241 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     6301 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     6361 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     6421 ggcgtagagg atcgagatcg atctcgatcc cgcgaaat
//
