LOCUS       sequence-322527-        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-HB1lock-L340W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(43..48,49..65,66..72)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    80..96
                     /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     85..107
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    113..182
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             233..259
                     /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             275..292
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             296..328
                     /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(332..340,341..355)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1916..1933)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2086..2172
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2264..2291
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2310..2401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2402..2470,2471..3262)
                     /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(2620..2639)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3433..4021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3922..3941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4175..4192
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4207..4347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4273..4292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4433..4455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4533..4610
                     /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             4611..5693
                     /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(4630..4649)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatccg
       61 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagcgccgct
      121 gagaaaaagc gaagcggcac tgctctttaa caatttatca gacaatctgt gtgggcactc
      181 gaccggaatt atcgattaac tttattatta aaaattaaag aggtatatat taatgtatcg
      241 attaaataag gaggaataaa ccatgggggg ttctcatcat catcatcatc atggtatggc
      301 tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataaggatcg
      361 atggggatcc gaaaccccga cgaaaccgaa agcggcccag accgaaaaga aaaccgagaa
      421 aaaaccggaa aacagcaatt ctgaagcagc taaaaaagcg ctgaacgatt atatttgggg
      481 cctgcagtac gacaaactga acatcctgac ccatcaaggt gaaaaactga aaaatcatag
      541 ctctcgtgaa gcctttcacc gtccgggcga atacgtggtt atcgaaaaga aaaaacagag
      601 tatctccaac gcgacctcga aactgagcgt gagttccgcc aatgatgacc gtatttttcc
      661 gggcgcgctg ctgaaagctg atcaaagcct gctggaaaac ctgccgaccc tgattccggt
      721 caatcgcggt aaaaccacga tctcagtgaa cctgccgggc ctgaaaaacg gtgaatcgaa
      781 tctgacggtc gaaaacccgt caaattcgac cgtgcgtacg gcggttaaca atctggtcga
      841 aaaatggatt cagaactaca gtaaaaccca tgcagtgccg gctcgcatgc agtacgaaag
      901 catctctgca caaagtatgt cccagctgca atgcaaattt ggcgccgatt tcagcaaagt
      961 gggtgctccg ctgaacgtgg acttttcatc ggttcacaaa ggcgaaaaac aggttttcat
     1021 cgcaaacttc cgtcaagttt actacaccgc ttctgtcgat agtccgaaca gcccgtcggc
     1081 gctgttcggc agtggtatta ccccgacgga cctgatcaac cgtggcgtta attccaaaac
     1141 cccgccggtc tatgtgtcca atgtgagcta tggccgcgcg atgtacgtta aatttgaaac
     1201 cacgagcaaa tctaccaaag tgcaggcggc catcgatgcc gtcgtgaaag gcgcaaaact
     1261 gaaagctggt acggaatacg aaaacatctg gaaaaacacc aaaatcacgg cagttgtcct
     1321 gggcggtaat ccgggcgaag ccagcaaagt tattaccggt aatatcgata cgctgaaaga
     1381 cctgattcag aaaggttcga actttagcgc gcaatctccg gccgttccga tcagttatac
     1441 cacgtccttc gtcaaagata atagcattgc aaccatccag tgcaatacgg actatattga
     1501 aaccaaagtg acgtcttaca aagatggcgc tctgaccctg aaccatgacg gtgcgtttgt
     1561 cgcccgcttc tatgtgtact gggaagaact gggccacgat gcagacggtt atgaaaccat
     1621 tcgtagtcgc tcctggtcag gcaacggtta taatcgtggc gcccattaca gcaccacgct
     1681 gcgctttaag ggtaacgttc gtaatatccg cgttaaagtc ctgggcgcaa ccggtctggc
     1741 ttgggaaccg tggcgtctga tttacagcaa aaacgatctg ccgctggtgc cgcagcgcaa
     1801 tatctctacc tggggcacca cgctgcaccc gcaattcgaa gataaagtgg ttaaagataa
     1861 cacggactaa gaattcgaag cttggctgtt ttggcggatg agagaagatt ttcagcctga
     1921 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     1981 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     2041 gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     2101 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2161 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     2221 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     2281 gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat
     2341 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2401 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2461 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2521 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2581 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2641 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2701 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2761 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2821 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2881 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2941 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3001 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3061 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3121 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3181 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3241 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3301 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3361 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3421 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3541 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4021 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4081 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4141 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4201 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     4261 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4321 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4381 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4441 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca gatcaattcg
     4501 cgcgcgaagg cgaagcggca tgcatttacg ttgacaccat cgaatggtgc aaaacctttc
     4561 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     4621 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     4681 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     4741 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     4801 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     4861 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     4921 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     4981 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     5041 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     5101 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     5161 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     5221 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     5281 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     5341 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     5401 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     5461 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     5521 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     5581 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5641 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5701 gcaattaatg taagttagcg cgaattgatc tggtttgaca gcttatcatc gactgcacgg
     5761 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     5821 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     5881 cg
//
