LOCUS       sequence-446165-        5210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Block V Repeats-In-Toxin (RTX) protein with global substitution of 
            alanine in nonapeptide repeats and C-terminal Capping Domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5210)
  AUTHORS   Chang M, Huang W, Shambharkar G, Hernandez K, Mai DJ
  TITLE     Sequence-defined structural transitions by calcium-responsive 
            proteins
  JOURNAL   Polym Chem. 2024. doi: https://doi.org/10.1039/D4PY00907J
REFERENCE   2  (bases 1 to 5210)
  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: "Polym Chem.
            2024. doi: https://doi.org/10.1039/D4PY00907J"
COMMENT     Sequence Label: pQE-9-RTXWTM-X2A-C
FEATURES             Location/Qualifiers
     source          1..5210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      667..761
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             805..1464
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(871..890)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1529..1615
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1708..2790)
                     /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     2752..2771
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2791..2868)
                     /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(2952..2974)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3060..3200
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3115..3134
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3215..3232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3386..3974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3466..3485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4145..4936,4937..5005))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4768..4787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5006..5110)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5178..5196
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc catatggagc tcggcgctag cggtagtgcg
      181 cgcgcggatg ttctgatagg cgatgcgggt gcaaatgtgc tgaacgggtt agccggtgca
      241 gacgttctgt ctggtggagc aggagccgac gttcttctcg gcgatgaggg cgccgatctg
      301 ttatcaggtg acgccggggc cgacgatctg tttgggggcc agggagccga tacgtacttg
      361 ttcggcgtgg gctacggtgc ggacaccatc tatgaaagcg gtggaggtgc agataccatt
      421 cgtatcaacg ctggagcaga tcagctctgg tttgctcgcc agggcaatga tcttgaaatt
      481 cgtatattgg ggactgacga tgctctgaca gtacatgatt ggtatagaga tgcggatcac
      541 cgggtcgaaa ttattcatgc tgcaaatcag gcagtagatc aagcgggtat cgaaaaatta
      601 gtcgaggcca tggctcaata tccggacgaa ttcactagtc tcgagaagct taattagctg
      661 agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca
      721 gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag cttggcgaga
      781 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
      841 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
      901 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
      961 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     1021 tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     1081 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     1141 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     1201 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     1261 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     1321 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     1381 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     1441 gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct
     1501 ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt cgaaagactg
     1561 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     1621 ctctagatta cgtgcagtcg atgataagct gtcaaacatg agaattgtgc ctaatgagtg
     1681 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1801 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     1861 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     1921 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     1981 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     2041 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     2101 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     2161 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     2221 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     2281 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     2341 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     2401 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     2461 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     2521 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     2581 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     2641 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     2701 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     2761 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     2821 gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg aatttcgggc
     2881 agcgttgggt cctggccacg ggtgcgcatg atctagagct gcctcgcgcg tttcggtgat
     2941 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3001 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3061 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     3121 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     3181 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3241 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3301 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3361 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3421 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3481 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3541 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3601 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3661 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3721 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3781 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3841 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3901 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3961 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4021 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4081 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4141 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4201 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4261 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4321 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4381 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4441 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4501 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4561 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4621 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4681 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4741 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4801 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4861 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4921 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4981 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5041 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5101 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     5161 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
//
