LOCUS       sequence-322545-        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-charge+
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(87..92,93..109,110..116)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    124..140
                     /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     129..151
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    157..226
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             277..303
                     /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             319..336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             340..372
                     /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(376..384,385..399)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1960..1977)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2130..2216
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2308..2335
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2354..2445
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2446..2514,2515..3306)
                     /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(2664..2683)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3477..4065
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3966..3985
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4219..4236
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4251..4391
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4317..4336)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4477..4499
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4577..4654
                     /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             4655..5737
                     /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(4674..4693)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt
       61 tctggcaaat attctgaaat gagctgttga caattaatca tccggctcgt ataatgtgtg
      121 gaattgtgag cggataacaa tttcacacag gaaacagcgc cgctgagaaa aagcgaagcg
      181 gcactgctct ttaacaattt atcagacaat ctgtgtgggc actcgaccgg aattatcgat
      241 taactttatt attaaaaatt aaagaggtat atattaatgt atcgattaaa taaggaggaa
      301 taaaccatgg ggggttctca tcatcatcat catcatggta tggctagcat gactggtgga
      361 cagcaaatgg gtcgggatct gtacgacgat gacgataagg atcgatgggg atccgaaacc
      421 ccgacgaaac cgaaagcggc ccagaccgaa aagaaaaccg agaaaaaacc ggaaaacagc
      481 aattctgaag cagctaaaaa agcgctgaac gattatattt ggggcctgca gtacgacaaa
      541 ctgaacatcc tgacccatca aggtgaaaaa ctgaaaaatc atagctctcg tgaagccttt
      601 caccgtccgg gcgaatacgt ggttatcgaa aagaaaaaac agagtatctc caacgcgacc
      661 tcgaaactga gcgtgagttc cgccaatgat gaccgtattt ttccgggcgc gctgctgaaa
      721 gctgatcaaa gcctgctgga aaacctgccg accctgattc cggtcaatcg cggtaaaacc
      781 acgatctcag tgaacctgcc gggcctgaaa aacggtgaat cgaatctgac ggtcgaaaac
      841 ccgtcaaatt cgaccgtgcg tacggcggtt aacaatctgg tcgaaaaatg gattcagaac
      901 tacagtaaaa cccatgcagt gccggctcgc atgcagtgcg aaagcatctc tgcacaaagt
      961 atgtcccagc tgcaagcgaa atttggcgcc gatttcagca aagtgggtgc tccgctgaac
     1021 gtggactttt catcggttca caaaggcgaa aaacaggttt tcatcgcaaa cttccgtcaa
     1081 gtttactaca ccgcttctgt cgatagtccg aacagcccgt cggcgctgtt cggcagtggt
     1141 attaccccga cggacctgat caaccgtggc gttaattcca aaaccccgcc ggtctatgtg
     1201 tccaatgtga gctatggccg cgcgatgtac gttaaatttg aaaccacgag caaatctacc
     1261 aaagtgcagg cggccatctg tgccgtcgtg aaaggcgcaa aactgaaagc tggtacggaa
     1321 aaggaaaaca tcaagaaaaa caccaaaatc acggcagttg tcctgggcgg taatccgggc
     1381 gaagccagca aagttattac cggtaatatc gatacgctga aagacctgat tcagaaaggt
     1441 tcgaacttta gcgcgcaatc tccggccgtt ccgatcagtt ataccacgtc cttcgtcaaa
     1501 gataatagca ttgcaaccat ccagaacaat acggactata ttgaaaccaa agtgacgtct
     1561 tacaaagatg gcgctctgac cctgaaccat gacggtgcgt ttgtcgcccg cttctatgtg
     1621 tactgggaag aactgggcca cgatgcagac ggttatgaaa ccattcgtag tcgctcctgg
     1681 tcaggcaacg gttataatcg tggcgcccat tacagcacca cgctgcgctt taagggtaac
     1741 gttcgtaata tccgcgttaa agtcctgggc gcaaccggtc tggcttggga accgtggcgt
     1801 ctgatttaca gcaaaaacga tctgccgctg gtgccgcagc gcaatatctc tacctggggc
     1861 accacgctgc acccgcaatt cgaagataaa gtggttaaag ataacacgga ctaagaattc
     1921 gaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     1981 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     2041 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     2101 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     2161 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2221 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     2281 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     2341 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2401 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2461 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2521 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2581 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2641 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     2701 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2761 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2821 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2881 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2941 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3001 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3061 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3121 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3181 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3241 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3301 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3361 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3421 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3481 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3541 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3601 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3661 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3721 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3781 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3841 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3901 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3961 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4021 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4081 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4141 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4201 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4261 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     4321 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4381 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4441 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4501 tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc
     4561 ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     4621 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     4681 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     4741 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     4801 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     4861 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     4921 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     4981 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5041 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     5101 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     5161 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     5221 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     5281 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     5341 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     5401 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     5461 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     5521 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     5581 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     5641 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5701 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     5761 agcgcgaatt gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg
     5821 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
     5881 tc
//
