LOCUS       sequence-322463-        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
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(129..134,135..151,152..158)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    166..182
                     /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     171..193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    199..268
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             319..345
                     /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             361..378
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             382..414
                     /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(418..426,427..441)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(2002..2019)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2172..2258
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2350..2377
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2396..2487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2488..2556,2557..3348)
                     /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(2706..2725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3519..4107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4008..4027
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4261..4278
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4293..4433
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4359..4378)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4519..4541
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4619..4696
                     /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             4697..5779
                     /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(4716..4735)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
       61 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
      121 atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg agcggataac
      181 aatttcacac aggaaacagc gccgctgaga aaaagcgaag cggcactgct ctttaacaat
      241 ttatcagaca atctgtgtgg gcactcgacc ggaattatcg attaacttta ttattaaaaa
      301 ttaaagaggt atatattaat gtatcgatta aataaggagg aataaaccat ggggggttct
      361 catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat
      421 ctgtacgacg atgacgataa ggatcgatgg ggatccgaaa ccccgacgaa accgaaagcg
      481 gcccagaccg aaaagaaaac cgagaaaaaa ccggaaaaca gcaattctga agcagctaaa
      541 aaagcgctga acgattatat ttggggcctg cagtacgaca aactgaacat cctgacccat
      601 caaggtgaaa aactgaaaaa tcatagctct cgtgaagcct ttcaccgtcc gggcgaatac
      661 gtggttatcg aaaagaaaaa acagagtatc tccaacgcga cctcgaaact gagcgtgagt
      721 tccgccaatg atgaccgtat ttttccgggc gcgctgctga aagctgatca aagcctgctg
      781 gaaaacctgc cgaccctgat tccggtcaat cgcggtaaaa ccacgatctc agtgaacctg
      841 ccgggcctga aaaacggtga atcgaatctg acggtcgaaa acccgtcaaa ttcgaccgtg
      901 cgtacggcgg ttaacaatct ggtcgaaaaa tggattcaga actacagtaa aacccatgca
      961 gtgccggctc gcatgcagta cgaaagcatc tctgcacaaa gtatgtccca gctgcaatgc
     1021 aaatttggcg ccgatttcag caaagtgggt gctccgctga acgtggactt ttcatcggtt
     1081 cacaaaggcg aaaaacaggt tttcatcgca aacttccgtc aagtttacta caccgcttct
     1141 gtcgatagtc cgaacagccc gtcggcgctg ttcggcagtg gtattacccc gacggacctg
     1201 atcaaccgtg gcgttaattc caaaaccccg ccggtctatg tgtccaatgt gagctatggc
     1261 cgcgcgatgt acgttaaatt tgaaaccacg agcaaatcta ccaaagtgca ggcggccatc
     1321 gatgccgtcg tgaaaggcgc aaaactgaaa gctggtacgg aatacgaaaa catcctgaaa
     1381 aacaccaaaa tcacggcagt tgtcctgggc ggtaatccgg gcgaagccag caaagttatt
     1441 accggtaata tcgatacgct gaaagacctg attcagaaag gttcgaactt tagcgcgcaa
     1501 tctccggccg ttccgatcag ttataccacg tccttcgtca aagataatag cattgcaacc
     1561 atccagtgca atacggacta tattgaaacc aaagtgacgt cttacaaaga tggcgctctg
     1621 accctgaacc atgacggtgc gtttgtcgcc cgcttctatg tgtactggga agaactgggc
     1681 cacgatgcag acggttatga aaccattcgt agtcgctcct ggtcaggcaa cggttataat
     1741 cgtggcgccc attacagcac cacgctgcgc tttaagggta acgttcgtaa tatccgcgtt
     1801 aaagtcctgg gcgcaaccgg tctggcttgg gaaccgtggc gtctgattta cagcaaaaac
     1861 gatctgccgc tggtgccgca gcgcaatatc tctacctggg gcaccacgct gcacccgcaa
     1921 ttcgaagata aagtggttaa agataacacg gactaagaat tcgaagcttg gctgttttgg
     1981 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     2041 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     2101 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     2161 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     2221 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     2281 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     2341 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     2401 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2461 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2521 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2581 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2641 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2701 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     2761 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2821 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2881 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2941 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3001 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3061 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3121 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3181 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3241 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3301 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3361 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3421 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3481 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3541 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3601 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3661 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3721 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3781 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3841 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3901 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3961 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4021 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4081 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4141 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4201 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4261 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4321 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     4381 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4441 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4501 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4561 aacgcgcgag gcagcagatc aattcgcgcg cgaaggcgaa gcggcatgca tttacgttga
     4621 caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4681 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4741 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4801 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4861 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4921 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4981 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     5041 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     5101 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     5161 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     5221 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     5281 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     5341 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     5401 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5461 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5521 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5581 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5641 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     5701 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5761 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagcgcgaa ttgatctggt
     5821 ttgacagctt atcatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg
     5881 aa
//
