LOCUS       sequence-322544-        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
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(154..159,160..176,177..183)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    191..207
                     /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     196..218
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    224..293
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             344..370
                     /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             386..403
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             407..439
                     /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(443..451,452..466)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2027..2044)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2197..2283
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2375..2402
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2421..2512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2513..2581,2582..3373)
                     /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(2731..2750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3544..4132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4033..4052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4286..4303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4318..4458
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4384..4403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4544..4566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4644..4721
                     /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             4722..5804
                     /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(4741..4760)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
       61 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
      121 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata
      181 atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagcgccgc tgagaaaaag
      241 cgaagcggca ctgctcttta acaatttatc agacaatctg tgtgggcact cgaccggaat
      301 tatcgattaa ctttattatt aaaaattaaa gaggtatata ttaatgtatc gattaaataa
      361 ggaggaataa accatggggg gttctcatca tcatcatcat catggtatgg ctagcatgac
      421 tggtggacag caaatgggtc gggatctgta cgacgatgac gataaggatc gatggggatc
      481 cgaaaccccg acgaaaccga aagcggccca gaccgaaaag aaaaccgaga aaaaaccgga
      541 aaacagcaat tctgaagcag ctaaaaaagc gctgaacgat tatatttggg gcctgcagta
      601 cgacaaactg aacatcctga cccatcaagg tgaaaaactg aaaaatcata gctctcgtga
      661 agcctttcac cgtccgggcg aatacgtggt tatcgaaaag aaaaaacaga gtatctccaa
      721 cgcgacctcg aaactgagcg tgagttccgc caatgatgac cgtatttttc cgggcgcgct
      781 gctgaaagct gatcaaagcc tgctggaaaa cctgccgacc ctgattccgg tcaatcgcgg
      841 taaaaccacg atctcagtga acctgccggg cctgaaaaac ggtgaatcga atctgacggt
      901 cgaaaacccg tcaaattcga ccgtgcgtac ggcggttaac aatctggtcg aaaaatggat
      961 tcagaactac agtaaaaccc atgcagtgcc ggctcgcatg cagtgcgaaa gcatctctgc
     1021 acaaagtatg tcccagctgc aagcgaaatt tggcgccgat ttcagcaaag tgggtgctcc
     1081 gctgaacgtg gacttttcat cggttcacaa aggcgaaaaa caggttttca tcgcaaactt
     1141 ccgtcaagtt tactacaccg cttctgtcga tagtccgaac agcccgtcgg cgctgttcgg
     1201 cagtggtatt accccgacgg acctgatcaa ccgtggcgtt aattccaaaa ccccgccggt
     1261 ctatgtgtcc aatgtgagct atggccgcgc gatgtacgtt aaatttgaaa ccacgagcaa
     1321 atctaccaaa gtgcaggcgg ccatctgtgc cgtcgtgaaa ggcgcaaaac tgaaagctgg
     1381 tacggaatac gaaaacatcc tgaaaaacac caaaatcacg gcagttgtcc tgggcggtaa
     1441 tccgggcgaa gccagcaaag ttattaccgg taatatcgat acgctgaaag acctgattca
     1501 gaaaggttcg aactttagcg cgcaatctcc ggccgttccg atcagttata ccacgtcctt
     1561 cgtcaaagat aatagcattg caaccatcca gaacaatacg gactatattg aaaccaaagt
     1621 gacgtcttac aaagatggcg ctctgaccct gaaccatgac ggtgcgtttg tcgcccgctt
     1681 ctatgtgtac tgggaagaac tgggccacga tgcagacggt tatgaaacca ttcgtagtcg
     1741 ctcctggtca ggcaacggtt ataatcgtgg cgcccattac agcaccacgc tgcgctttaa
     1801 gggtaacgtt cgtaatatcc gcgttaaagt cctgggcgca accggtctgg cttgggaacc
     1861 gtggcgtctg atttacagca aaaacgatct gccgctggtg ccgcagcgca atatctctac
     1921 ctggggcacc acgctgcacc cgcaattcga agataaagtg gttaaagata acacggacta
     1981 agaattcgaa gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     2041 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     2101 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     2161 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     2221 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     2281 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     2341 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     2401 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct
     2461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     2761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3661 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3721 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3781 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3841 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3901 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3961 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4021 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4081 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4141 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4201 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4261 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4321 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     4381 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4441 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4501 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4561 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag
     4621 gcgaagcggc atgcatttac gttgacacca tcgaatggtg caaaaccttt cgcggtatgg
     4681 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     4741 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     4801 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     4861 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     4921 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     4981 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     5041 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     5101 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     5161 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     5221 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     5281 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     5341 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     5401 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     5461 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     5521 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     5581 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     5641 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     5701 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5761 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5821 gtaagttagc gcgaattgat ctggtttgac agcttatcat cgactgcacg gtgcaccaat
     5881 gc
//
