LOCUS       sequence-322569-        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-HB2lock-N342W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(72..77,78..94,95..101)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    109..125
                     /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     114..136
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    142..211
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             262..288
                     /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             304..321
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             325..357
                     /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(361..369,370..384)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1945..1962)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2115..2201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2293..2320
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2339..2430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2431..2499,2500..3291)
                     /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(2649..2668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3462..4050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3951..3970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4204..4221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4236..4376
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4302..4321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4462..4484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4562..4639
                     /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             4640..5722
                     /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(4659..4678)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
       61 gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt gtgagcggat
      121 aacaatttca cacaggaaac agcgccgctg agaaaaagcg aagcggcact gctctttaac
      181 aatttatcag acaatctgtg tgggcactcg accggaatta tcgattaact ttattattaa
      241 aaattaaaga ggtatatatt aatgtatcga ttaaataagg aggaataaac catggggggt
      301 tctcatcatc atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg
      361 gatctgtacg acgatgacga taaggatcga tggggatccg aaaccccgac gaaaccgaaa
      421 gcggcccaga ccgaaaagaa aaccgagaaa aaaccggaaa acagcaattc tgaagcagct
      481 aaaaaagcgc tgaacgatta tatttggggc ctgcagtacg acaaactgaa catcctgacc
      541 catcaaggtg aaaaactgaa aaatcatagc tctcgtgaag cctttcaccg tccgggcgaa
      601 tacgtggtta tcgaaaagaa aaaacagagt atctccaacg cgacctcgaa actgagcgtg
      661 agttccgcca atgatgaccg tatttttccg ggcgcgctgc tgaaagctga tcaaagcctg
      721 ctggaaaacc tgccgaccct gattccggtc aatcgcggta aaaccacgat ctcagtgaac
      781 ctgccgggcc tgaaaaacgg tgaatcgaat ctgacggtcg aaaacccgtc aaattcgacc
      841 gtgcgtacgg cggttaacaa tctggtcgaa aaatggattc agaactacag taaaacccat
      901 gcagtgccgg ctcgcatgca gtgcgaaagc atctctgcac aaagtatgtc ccagctgcaa
      961 gcgaaatttg gcgccgattt cagcaaagtg ggtgctccgc tgaacgtgga cttttcatcg
     1021 gttcacaaag gcgaaaaaca ggttttcatc gcaaacttcc gtcaagttta ctacaccgct
     1081 tctgtcgata gtccgaacag cccgtcggcg ctgttcggca gtggtattac cccgacggac
     1141 ctgatcaacc gtggcgttaa ttccaaaacc ccgccggtct atgtgtccaa tgtgagctat
     1201 ggccgcgcga tgtacgttaa atttgaaacc acgagcaaat ctaccaaagt gcaggcggcc
     1261 atctgtgccg tcgtgaaagg cgcaaaactg aaagctggta cggaatacga aaacatcctg
     1321 aaatggacca aaatcacggc agttgtcctg ggcggtaatc cgggcgaagc cagcaaagtt
     1381 attaccggta atatcgatac gctgaaagac ctgattcaga aaggttcgaa ctttagcgcg
     1441 caatctccgg ccgttccgat cagttatacc acgtccttcg tcaaagataa tagcattgca
     1501 accatccaga acaatacgga ctatattgaa accaaagtga cgtcttacaa agatggcgct
     1561 ctgaccctga accatgacgg tgcgtttgtc gcccgcttct atgtgtactg ggaagaactg
     1621 ggccacgatg cagacggtta tgaaaccatt cgtagtcgct cctggtcagg caacggttat
     1681 aatcgtggcg cccattacag caccacgctg cgctttaagg gtaacgttcg taatatccgc
     1741 gttaaagtcc tgggcgcaac cggtctggct tgggaaccgt ggcgtctgat ttacagcaaa
     1801 aacgatctgc cgctggtgcc gcagcgcaat atctctacct ggggcaccac gctgcacccg
     1861 caattcgaag ataaagtggt taaagataac acggactaag aattcgaagc ttggctgttt
     1921 tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     1981 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     2041 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     2101 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     2161 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     2221 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     2281 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     2341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2641 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     2701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2941 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3601 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4261 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     4321 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4381 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4441 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4501 cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt
     4561 tgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     4621 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     4681 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     4741 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     4801 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     4861 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     4921 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     4981 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     5041 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     5101 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     5161 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     5221 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     5281 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     5341 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     5401 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     5461 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     5521 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     5581 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     5641 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     5701 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc gaattgatct
     5761 ggtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat
     5821 cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg
     5881 cg
//
