LOCUS       sequence-446177-        5201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repeats-In-Toxin (RTX) protein consensus sequence GGAGEDTLY repeated
            nine times with C-terminal Capping Domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5201)
  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 5201)
  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-RTXMCR-TLY-X2E-C
FEATURES             Location/Qualifiers
     source          1..5201
                     /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      658..752
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             796..1455
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(862..881)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1520..1606
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1699..2781)
                     /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     2743..2762
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2782..2859)
                     /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(2943..2965)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3051..3191
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3106..3125
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3206..3223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3377..3965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3457..3476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4136..4927,4928..4996))
                     /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     4759..4778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4997..5101)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5169..5187
                     /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 cggcggtgca
      181 ggtgaagata ctctctacgg cggtgcgggt gaggatacct tgtacggtgg agccggtgag
      241 gacacattgt acggtggcgc aggtgaagac acattgtatg gcggtgctgg cgaagacacg
      301 ctctatggtg gtgctggtga ggacacgctt tacggtggtg caggcgagga caccctgtac
      361 ggtggtgccg gggaagacac tctttacggt ggagccggcg aggacactct ttacataaat
      421 gccggcgctg accagttgtg gtttgcgcgc caaggaaatg atcttgaaat acgcatctta
      481 ggaaccgacg acgctttaac cgtccatgat tggtaccgcg acgcggacca tcgtgttgag
      541 atcattcacg ctgcgaacca agctgttgat caagccggga ttgagaagct ggtggaagca
      601 atggcccaat acccggatga attcactagt ctcgagaagc ttaattagct gagcttggac
      661 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
      721 gttgccgccg ggcgtttttt attggtgaga atccaagcta gcttggcgag attttcagga
      781 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
      841 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
      901 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
      961 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg
     1021 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     1081 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     1141 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     1201 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     1261 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
     1321 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     1381 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     1441 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg
     1501 actctctagc ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1561 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cctctagatt
     1621 acgtgcagtc gatgataagc tgtcaaacat gagaattgtg cctaatgagt gagctaactt
     1681 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1741 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     1801 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     1861 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     1921 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     1981 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     2041 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     2101 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     2161 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     2221 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     2281 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     2341 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     2401 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     2461 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     2521 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     2581 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     2641 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     2701 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     2761 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     2821 tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gaatttcggg cagcgttggg
     2881 tcctggccac gggtgcgcat gatctagagc tgcctcgcgc gtttcggtga tgacggtgaa
     2941 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3001 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3061 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3121 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3181 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3241 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3301 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3361 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3421 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3481 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3541 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3601 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3661 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3721 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3781 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     3841 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3901 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3961 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4021 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4081 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4141 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4201 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4261 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4321 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4381 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4441 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4501 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4561 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4621 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4681 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4741 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4801 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4861 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4921 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4981 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5041 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5101 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     5161 cctataaaaa taggcgtatc acgaggccct ttcgtcttca c
//
