LOCUS       sequence-322568-        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-L340W
FEATURES             Location/Qualifiers
     source          1..5882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(554..1345,1346..1414))
                     /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     1177..1196
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1415..1506)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(1525..1552)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1644..1730)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1883..1900
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(join(3461..3475,3476..3484))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(3488..3520)
                     /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             complement(3524..3541)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3557..3583)
                     /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"
     misc_feature    3634..3703
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     primer_bind     complement(3709..3731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3720..3736
                     /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)."
     promoter        complement(join(3744..3750,3751..3767,3768..3773))
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(4005..5087)
                     /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     5049..5068
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5088..5165)
                     /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."
     primer_bind     complement(5243..5265)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5351..5491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5406..5425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5506..5523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5677..5882,1..383))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5757..5776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
       61 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      121 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      181 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
      241 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
      301 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
      361 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
      421 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
      481 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
      541 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
      601 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
      661 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
      721 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
      781 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
      841 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
      901 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
      961 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1021 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1081 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1141 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     1201 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1261 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1321 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     1381 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     1441 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1501 aacaaaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt
     1561 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcgca
     1621 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     1681 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggca
     1741 gttccctact ctcgcatggg gagaccccac actaccatcg gcgctacggc gtttcacttc
     1801 tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca aattctgttt
     1861 tatcagaccg cttctgcgtt ctgatttaat ctgtatcagg ctgaaaatct tctctcatcc
     1921 gccaaaacag ccaagcttcg aattcttagt ccgtgttatc tttaaccact ttatcttcga
     1981 attgcgggtg cagcgtggtg ccccaggtag agatattgcg ctgcggcacc agcggcagat
     2041 cgtttttgct gtaaatcaga cgccacggtt cccaagccag accggttgcg cccaggactt
     2101 taacgcggat attacgaacg ttacccttaa agcgcagcgt ggtgctgtaa tgggcgccac
     2161 gattataacc gttgcctgac caggagcgac tacgaatggt ttcataaccg tctgcatcgt
     2221 ggcccagttc ttcccagtac acatagaagc gggcgacaaa cgcaccgtca tggttcaggg
     2281 tcagagcgcc atctttgtaa gacgtcactt tggtttcaat atagtccgta ttgttctgga
     2341 tggttgcaat gctattatct ttgacgaagg acgtggtata actgatcgga acggccggag
     2401 attgcgcgct aaagttcgaa cctttctgaa tcaggtcttt cagcgtatcg atattaccgg
     2461 taataacttt gctggcttcg cccggattac cgcccaggac aactgccgtg attttggtgt
     2521 ttttccagat gttttcgtat tccgtaccag ctttcagttt tgcgcctttc acgacggcac
     2581 agatggccgc ctgcactttg gtagatttgc tcgtggtttc aaatttaacg tacatcgcgc
     2641 ggccatagct cacattggac acatagaccg gcggggtttt ggaattaacg ccacggttga
     2701 tcaggtccgt cggggtaata ccactgccga acagcgccga cgggctgttc ggactatcga
     2761 cagaagcggt gtagtaaact tgacggaagt ttgcgatgaa aacctgtttt tcgcctttgt
     2821 gaaccgatga aaagtccacg ttcagcggag cacccacttt gctgaaatcg gcgccaaatt
     2881 tcgcttgcag ctgggacata ctttgtgcag agatgctttc gcactgcatg cgagccggca
     2941 ctgcatgggt tttactgtag ttctgaatcc atttttcgac cagattgtta accgccgtac
     3001 gcacggtcga atttgacggg ttttcgaccg tcagattcga ttcaccgttt ttcaggcccg
     3061 gcaggttcac tgagatcgtg gttttaccgc gattgaccgg aatcagggtc ggcaggtttt
     3121 ccagcaggct ttgatcagct ttcagcagcg cgcccggaaa aatacggtca tcattggcgg
     3181 aactcacgct cagtttcgag gtcgcgttgg agatactctg ttttttcttt tcgataacca
     3241 cgtattcgcc cggacggtga aaggcttcac gagagctatg atttttcagt ttttcacctt
     3301 gatgggtcag gatgttcagt ttgtcgtact gcaggcccca aatataatcg ttcagcgctt
     3361 ttttagctgc ttcagaattg ctgttttccg gttttttctc ggttttcttt tcggtctggg
     3421 ccgctttcgg tttcgtcggg gtttcggatc cccatcgatc cttatcgtca tcgtcgtaca
     3481 gatcccgacc catttgctgt ccaccagtca tgctagccat accatgatga tgatgatgat
     3541 gagaaccccc catggtttat tcctccttat ttaatcgata cattaatata tacctcttta
     3601 atttttaata ataaagttaa tcgataattc cggtcgagtg cccacacaga ttgtctgata
     3661 aattgttaaa gagcagtgcc gcttcgcttt ttctcagcgg cgctgtttcc tgtgtgaaat
     3721 tgttatccgc tcacaattcc acacattata cgagccggat gattaattgt caacagctca
     3781 tttcagaata tttgccagaa ccgttatgat gtcggcgcaa aaaacattat ccagaacggg
     3841 agtgcgcctt gagcgacacg aattatgcag tgatttacga cctgcacagc cataccacag
     3901 cttccgatgg ctgcctgacg ccagaagcat tggtgcaccg tgcagtcgat gataagctgt
     3961 caaaccagat caattcgcgc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4141 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4201 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4261 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4321 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4381 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4441 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4501 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4561 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     4621 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     4681 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     4741 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     4801 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     4861 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4921 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4981 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5041 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5101 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     5161 gtgtcaacgt aaatgcatgc cgcttcgcct tcgcgcgcga attgatctgc tgcctcgcgc
     5221 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     5281 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     5341 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     5401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     5461 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     5521 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5581 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5641 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5701 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5761 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5821 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5881 ct
//
