LOCUS       sequence-322459-        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-prepore-N342C
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(81..86,87..103,104..110)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    118..134
                     /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     123..145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    151..220
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             271..297
                     /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             313..330
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             334..366
                     /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(370..378,379..393)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(1954..1971)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2124..2210
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2302..2329
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2348..2439
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2440..2508,2509..3300)
                     /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(2658..2677)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3471..4059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3960..3979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4213..4230
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4245..4385
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4311..4330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4471..4493
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4571..4648
                     /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             4649..5731
                     /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(4668..4687)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
       61 aaatattctg aaatgagctg ttgacaatta atcatccggc tcgtataatg tgtggaattg
      121 tgagcggata acaatttcac acaggaaaca gcgccgctga gaaaaagcga agcggcactg
      181 ctctttaaca atttatcaga caatctgtgt gggcactcga ccggaattat cgattaactt
      241 tattattaaa aattaaagag gtatatatta atgtatcgat taaataagga ggaataaacc
      301 atggggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
      361 atgggtcggg atctgtacga cgatgacgat aaggatcgat ggggatccga aaccccgacg
      421 aaaccgaaag cggcccagac cgaaaagaaa accgagaaaa aaccggaaaa cagcaattct
      481 gaagcagcta aaaaagcgct gaacgattat atttggggcc tgcagtacga caaactgaac
      541 atcctgaccc atcaaggtga aaaactgaaa aatcatagct ctcgtgaagc ctttcaccgt
      601 ccgggcgaat acgtggtttg cgaaaagaaa aaacagagta tctccaacgc gacctcgaaa
      661 ctgagcgtga gttccgccaa tgatgaccgt atttttccgg gcgcgctgct gaaagctgat
      721 caaagcctgc tggaaaacct gccgaccctg attccggtca atcgcggtaa aaccacgatc
      781 tcagtgaacc tgccgggcct gaaaaacggt gaatcgaatc tgacggtcga aaacccgtca
      841 aattcgaccg tgcgtacggc ggttaacaat ctggtcgaaa aatggattca gaactacagt
      901 aaaacccatg cagtgccggc tcgcatgcag tacgaaagca tctctgcaca aagtatgtcc
      961 cagctgcaag cgaaatttgg cgccgatttc agcaaagtgg gtgctccgct gaacgtggac
     1021 ttttcatcgg ttcacaaatg cgaaaaacag gttttcatcg caaacttccg tcaagtttac
     1081 tacaccgctt ctgtcgatag tccgaacagc ccgtcggcgc tgttcggcag tggtattacc
     1141 ccgacggacc tgatcaaccg tggcgttaat tccaaaaccc cgccggtcta tgtgtccaat
     1201 gtgagctatg gccgcgcgat gtacgttaaa tttgaaacca cgagcaaatc taccaaagtg
     1261 caggcggcca tcgatgccgt cgtgaaaggc gcaaaactga aagctggtac ggaatacgaa
     1321 aacatcctga aatgcaccaa aatcacggca gttgtcctgg gcggtaatcc gggcgaagcc
     1381 agcaaagtta ttaccggtaa tatcgatacg ctgaaagacc tgattcagaa aggttcgaac
     1441 tttagcgcgc aatctccggc cgttccgatc agttatacca cgtccttcgt caaagataat
     1501 agcattgcaa ccatccagaa caatacggac tatattgaaa ccaaagtgac gtcttacaaa
     1561 gatggcgctc tgaccctgaa ccatgacggt gcgtttgtcg cccgcttcta tgtgtactgg
     1621 gaagaactgg gccacgatgc agacggttat gaaaccattc gtagtcgctc ctggtcaggc
     1681 aacggttata atcgtggcgc ccattacagc accacgctgc gctttaaggg taacgttcgt
     1741 aatatccgcg ttaaagtcct gggcgcaacc ggtctggctt gggaaccgtg gcgtctgatt
     1801 tacagcaaaa acgatctgcc gctggtgccg cagcgcaata tctctacctg gggcaccacg
     1861 ctgcacccgc aattcgaaga taaagtggtt aaagataaca cggactaaga attcgaagct
     1921 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     1981 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     2041 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
     2101 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     2161 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     2221 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     2281 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     2341 aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2401 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2461 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2521 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2581 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2641 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     2701 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2761 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2821 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2881 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2941 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3001 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3061 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3121 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3181 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3241 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3301 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3361 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3421 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3481 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3541 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3601 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3661 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3721 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3781 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3841 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3901 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3961 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4021 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4081 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4141 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4201 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4261 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4321 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4381 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4441 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4501 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg
     4561 catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     4621 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     4681 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     4741 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     4801 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     4861 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     4921 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     4981 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5041 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     5101 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     5161 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     5221 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     5281 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     5341 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     5401 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     5461 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     5521 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     5581 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     5641 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5701 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg
     5761 aattgatctg gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
     5821 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     5881 cg
//
