LOCUS       sequence-322451-        5882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of inserted ILY variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5882)
  AUTHORS   Shah NR, Voisin TB, Parsons ES, Boyd CM, Hoogenboom BW, Bubeck D
  TITLE     Structural basis for tuning activity and membrane specificity of 
            bacterial cytolysins.
  JOURNAL   Nat Commun. 2020 Nov 16;11(1):5818. doi: 10.1038/s41467-020-19482-6.
  PUBMED    33199689
REFERENCE   2  (bases 1 to 5882)
  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: "Nat 
            Commun."; date: "2020-11-16"; volume: "11(1):5818. doi"; pages: " 
            10.1038/s41467-020-19482-6"
COMMENT     Sequence Label: pTricHis-ILY-WT-N342W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(342..347,348..364,365..371)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    379..395
                     /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     384..406
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    412..481
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             532..558
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             574..591
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             595..627
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(631..639,640..654)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2215..2232)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2385..2471
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2563..2590
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2609..2700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2701..2769,2770..3561)
                     /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(2919..2938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3732..4320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4221..4240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4474..4491
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4506..4646
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4572..4591)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4732..4754
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4832..4909
                     /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             join(4910..5882,1..110)
                     /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(4929..4948)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
       61 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      121 attaatgtaa gttagcgcga attgatctgg tttgacagct tatcatcgac tgcacggtgc
      181 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
      241 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
      301 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatccgg
      361 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agcgccgctg
      421 agaaaaagcg aagcggcact gctctttaac aatttatcag acaatctgtg tgggcactcg
      481 accggaatta tcgattaact ttattattaa aaattaaaga ggtatatatt aatgtatcga
      541 ttaaataagg aggaataaac catggggggt tctcatcatc atcatcatca tggtatggct
      601 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatcga
      661 tggggatccg aaaccccgac gaaaccgaaa gcggcccaga ccgaaaagaa aaccgagaaa
      721 aaaccggaaa acagcaattc tgaagcagct aaaaaagcgc tgaacgatta tatttggggc
      781 ctgcagtacg acaaactgaa catcctgacc catcaaggtg aaaaactgaa aaatcatagc
      841 tctcgtgaag cctttcaccg tccgggcgaa tacgtggtta tcgaaaagaa aaaacagagt
      901 atctccaacg cgacctcgaa actgagcgtg agttccgcca atgatgaccg tatttttccg
      961 ggcgcgctgc tgaaagctga tcaaagcctg ctggaaaacc tgccgaccct gattccggtc
     1021 aatcgcggta aaaccacgat ctcagtgaac ctgccgggcc tgaaaaacgg tgaatcgaat
     1081 ctgacggtcg aaaacccgtc aaattcgacc gtgcgtacgg cggttaacaa tctggtcgaa
     1141 aaatggattc agaactacag taaaacccat gcagtgccgg ctcgcatgca gtacgaaagc
     1201 atctctgcac aaagtatgtc ccagctgcaa gcgaaatttg gcgccgattt cagcaaagtg
     1261 ggtgctccgc tgaacgtgga cttttcatcg gttcacaaag gcgaaaaaca ggttttcatc
     1321 gcaaacttcc gtcaagttta ctacaccgct tctgtcgata gtccgaacag cccgtcggcg
     1381 ctgttcggca gtggtattac cccgacggac ctgatcaacc gtggcgttaa ttccaaaacc
     1441 ccgccggtct atgtgtccaa tgtgagctat ggccgcgcga tgtacgttaa atttgaaacc
     1501 acgagcaaat ctaccaaagt gcaggcggcc atcgatgccg tcgtgaaagg cgcaaaactg
     1561 aaagctggta cggaatacga aaacatcctg aaatggacca aaatcacggc agttgtcctg
     1621 ggcggtaatc cgggcgaagc cagcaaagtt attaccggta atatcgatac gctgaaagac
     1681 ctgattcaga aaggttcgaa ctttagcgcg caatctccgg ccgttccgat cagttatacc
     1741 acgtccttcg tcaaagataa tagcattgca accatccaga acaatacgga ctatattgaa
     1801 accaaagtga cgtcttacaa agatggcgct ctgaccctga accatgacgg tgcgtttgtc
     1861 gcccgcttct atgtgtactg ggaagaactg ggccacgatg cagacggtta tgaaaccatt
     1921 cgtagtcgct cctggtcagg caacggttat aatcgtggcg cccattacag caccacgctg
     1981 cgctttaagg gtaacgttcg taatatccgc gttaaagtcc tgggcgcaac cggtctggct
     2041 tgggaaccgt ggcgtctgat ttacagcaaa aacgatctgc cgctggtgcc gcagcgcaat
     2101 atctctacct ggggcaccac gctgcacccg caattcgaag ataaagtggt taaagataac
     2161 acggactaag aattcgaagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     2221 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     2281 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     2341 tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     2401 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     2461 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     2521 gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg
     2581 atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg
     2641 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2701 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2761 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2821 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2881 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2941 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3001 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3061 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3121 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3181 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3241 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3301 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3361 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3421 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3481 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3541 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3601 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3661 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3721 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3781 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3841 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3901 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3961 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4021 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4081 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4141 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4201 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4261 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4321 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4381 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4441 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4501 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     4561 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4621 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4681 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4741 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc
     4801 gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc gaatggtgca aaacctttcg
     4861 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     4921 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     4981 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     5041 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     5101 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     5161 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     5221 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     5281 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     5341 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     5401 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     5461 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     5521 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     5581 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     5641 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     5701 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     5761 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     5821 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     5881 ga
//
