LOCUS       sequence-322566-        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-N338W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(96..101,102..118,119..125)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    133..149
                     /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     138..160
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    166..235
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             286..312
                     /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             328..345
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             349..381
                     /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(385..393,394..408)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1969..1986)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2139..2225
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2317..2344
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2363..2454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2455..2523,2524..3315)
                     /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(2673..2692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3486..4074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3975..3994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4228..4245
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4260..4400
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4326..4345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4486..4508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4586..4663
                     /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             4664..5746
                     /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(4683..4702)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
       61 cataacggtt ctggcaaata ttctgaaatg agctgttgac aattaatcat ccggctcgta
      121 taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagcgcc gctgagaaaa
      181 agcgaagcgg cactgctctt taacaattta tcagacaatc tgtgtgggca ctcgaccgga
      241 attatcgatt aactttatta ttaaaaatta aagaggtata tattaatgta tcgattaaat
      301 aaggaggaat aaaccatggg gggttctcat catcatcatc atcatggtat ggctagcatg
      361 actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga tcgatgggga
      421 tccgaaaccc cgacgaaacc gaaagcggcc cagaccgaaa agaaaaccga gaaaaaaccg
      481 gaaaacagca attctgaagc agctaaaaaa gcgctgaacg attatatttg gggcctgcag
      541 tacgacaaac tgaacatcct gacccatcaa ggtgaaaaac tgaaaaatca tagctctcgt
      601 gaagcctttc accgtccggg cgaatacgtg gttatcgaaa agaaaaaaca gagtatctcc
      661 aacgcgacct cgaaactgag cgtgagttcc gccaatgatg accgtatttt tccgggcgcg
      721 ctgctgaaag ctgatcaaag cctgctggaa aacctgccga ccctgattcc ggtcaatcgc
      781 ggtaaaacca cgatctcagt gaacctgccg ggcctgaaaa acggtgaatc gaatctgacg
      841 gtcgaaaacc cgtcaaattc gaccgtgcgt acggcggtta acaatctggt cgaaaaatgg
      901 attcagaact acagtaaaac ccatgcagtg ccggctcgca tgcagtgcga aagcatctct
      961 gcacaaagta tgtcccagct gcaagcgaaa tttggcgccg atttcagcaa agtgggtgct
     1021 ccgctgaacg tggacttttc atcggttcac aaaggcgaaa aacaggtttt catcgcaaac
     1081 ttccgtcaag tttactacac cgcttctgtc gatagtccga acagcccgtc ggcgctgttc
     1141 ggcagtggta ttaccccgac ggacctgatc aaccgtggcg ttaattccaa aaccccgccg
     1201 gtctatgtgt ccaatgtgag ctatggccgc gcgatgtacg ttaaatttga aaccacgagc
     1261 aaatctacca aagtgcaggc ggccatctgt gccgtcgtga aaggcgcaaa actgaaagct
     1321 ggtacggaat acgaatggat cctgaaaaac accaaaatca cggcagttgt cctgggcggt
     1381 aatccgggcg aagccagcaa agttattacc ggtaatatcg atacgctgaa agacctgatt
     1441 cagaaaggtt cgaactttag cgcgcaatct ccggccgttc cgatcagtta taccacgtcc
     1501 ttcgtcaaag ataatagcat tgcaaccatc cagaacaata cggactatat tgaaaccaaa
     1561 gtgacgtctt acaaagatgg cgctctgacc ctgaaccatg acggtgcgtt tgtcgcccgc
     1621 ttctatgtgt actgggaaga actgggccac gatgcagacg gttatgaaac cattcgtagt
     1681 cgctcctggt caggcaacgg ttataatcgt ggcgcccatt acagcaccac gctgcgcttt
     1741 aagggtaacg ttcgtaatat ccgcgttaaa gtcctgggcg caaccggtct ggcttgggaa
     1801 ccgtggcgtc tgatttacag caaaaacgat ctgccgctgg tgccgcagcg caatatctct
     1861 acctggggca ccacgctgca cccgcaattc gaagataaag tggttaaaga taacacggac
     1921 taagaattcg aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
     1981 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
     2041 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
     2101 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
     2161 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     2221 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
     2281 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
     2341 tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg
     2401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2461 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2521 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2581 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2641 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2701 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2761 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2821 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2881 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2941 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     3001 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3061 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3121 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3181 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3241 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3301 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3361 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3421 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3481 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3601 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4141 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4201 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4261 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     4321 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4381 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4441 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4501 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga
     4561 aggcgaagcg gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat
     4621 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     4681 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4741 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4801 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4861 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     4921 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     4981 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     5041 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     5101 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     5161 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     5221 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     5281 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     5341 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     5401 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     5461 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     5521 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     5581 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     5641 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5701 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5761 atgtaagtta gcgcgaattg atctggtttg acagcttatc atcgactgca cggtgcacca
     5821 atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca
     5881 ct
//
