LOCUS       sequence-358071-        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) with C terminal cysteine for maleimide 
            chemistry.
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-E192C-del1-40
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    84..108
                     /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             124..144
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1133..1150
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1203..1221)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1217..1264
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1301..1756
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1388..1407)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1598..1619
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1782..1886
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1887..1955,1956..2747)
                     /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(2105..2124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2918..3506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3407..3426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3660..3677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3692..3831
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3757..3776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3917..3939
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3933..4124)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4933..6015)
                     /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     5977..5996
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6016..6093)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6299..6318
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
       61 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagtaata
      121 attttgttta actttaagaa ggaggtctag aatgggaggc agtaaaagtg aaagttctgc
      181 agctattcat gcaactgcta aatggtctac tgctcaatta aagaaaacac aggcagagca
      241 ggctgcccgg gcaaaagctg cagcggaagc acaggcgaaa gcaaaggcaa acagggatgc
      301 gctgactcag cgcctgaagg atatcgtaaa tgaggctctt cgtcacaatg cctcacgtac
      361 gccttcagca acagagcttg ctcatgctaa taatgcagct atgcaggcgg aagacgagcg
      421 tttgcgcctt gcgaaagcag aagaaaaagc ccgtaaagaa gcggaagcag cagaaaaggc
      481 ttttcaggaa gcagaacaac gacgtaaaga gattgaacgg gagaaggctg aaacagaacg
      541 ccagttgaaa ctggctgaag ctgaagagaa acgactggct gcattgagtg aagaagctaa
      601 agctgttgag atcgcccaaa aaaaactttc tgctgcacaa tctgaagtgg tgaaaatgga
      661 tggagagatt aagactctca attctcgttt aagctccagt atccatgccc gtgatgcaga
      721 aatgaaaacg ctcgctggaa aacgaaatga actggctcag gcatccgcta aatataaaga
      781 actggatgag ctggtcaaaa aactatcacc aagagccaat gatccgcttc agaaccgtcc
      841 tttttttgaa gcaaccagac gacgggttgg ggccggtaag attagagaag aaaaacaaaa
      901 acaggtaaca gcatcagaaa cacgtattaa ccggataaat gctgatataa ctcagatcca
      961 gaaggctatt tctcaggtca gtaataatcg taatgccggt atcgctcgtg ttcatgaagc
     1021 tgaagaaaat ttgaaaaaag cacagaataa tctccttaat tcacagatta aggatgctgt
     1081 tgatgcaaca gttagctttt atcaaacgct gactgaaaaa tatggtctct gccaccacca
     1141 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1201 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1261 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     1321 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1381 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1441 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1501 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1561 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1621 acaacactca accatatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1681 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1741 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1801 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1861 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1921 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1981 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2041 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2101 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2161 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2221 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2281 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2341 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2401 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     2461 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2521 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2581 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2641 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2701 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2761 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2821 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2881 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2941 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3001 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3061 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3121 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3181 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3241 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3301 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3361 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3421 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3481 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3541 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3601 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3661 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3721 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3781 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3841 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3901 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3961 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4021 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4081 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4141 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4201 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4261 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4321 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4381 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4441 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4501 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4561 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4621 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4681 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4741 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4801 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4861 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4921 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4981 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5041 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5101 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5161 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5221 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5281 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5341 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5401 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5461 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5521 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5581 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5641 gttaataatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5701 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5761 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5821 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5881 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5941 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6001 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6061 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6121 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6181 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6241 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6301 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc g
//
