LOCUS       sequence-360536-        4236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli plasmid encoding a fusion between pH-Countdown and the 
            calcium-binding domains of GECO 1.2 with a hexahistidine tag, 
            targeted to the periplasm via the TorA presequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4236)
  AUTHORS   Seidel ZP, Wang JCK, Riegler J, York AG, Ingaramo M
  TITLE     Relaxation Sensors
  JOURNAL   (2021), doi: 10.5281/zenodo.5810930
REFERENCE   2  (bases 1 to 4236)
  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: "(2021), 
            doi: 10.5281/zenodo.5810930"
COMMENT     Sequence Label: pRSET-TorA-6xHis-M13-pHCountdown-Calmodulin
FEATURES             Location/Qualifiers
     source          1..4236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..44
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             179..201
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             359..376
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(422..430,431..445)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             473..529
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(1746..1764)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1760..1807
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1904..2359
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1991..2010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2201..2222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2385..2489
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2490..2558,2559..3350)
                     /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     complement(2708..2727)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3521..4109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4010..4029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaatagcgcc
       61 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg atctcgatcc
      121 cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta gaaataattt
      181 tgtttaactt taagaaggag atatacatat gaataacaat gaccttttcc aagcatccag
      241 aagaaggttc ctagctcagc tgggttcttt gacagatgcg ggtactctag gtccaagttt
      301 attgactccc agaagggcta cagcagctca ggcagcaacg gatgcatcca ggggatcaca
      361 ccaccaccac caccacacta gtggaatggc atccatgaca ggcggtcaac agatgggaag
      421 agacttatat gatgacgacg acaaagactt ggctactatg gtagatagca gcagaaggaa
      481 atggaacaaa accgggcatg ctgttcgtgc cattggaaga ctgagctcag cggtgattaa
      541 accagacatg aagatcaagc tgcgtatgga aggctctgta aatggacacc gtttcagaat
      601 tgaaggagtt ggccttggga agcctcttga gggaaaacag agtatggacc ttaaagtcaa
      661 agaaggcgga cctctgcctt tcgcctatga catcttgaca atggcgttct gttacggtaa
      721 cagggtattc gccaaatacc cagaaaatat agtagactat ttcaagcagt cgtttcctga
      781 gggctactct tgggaacgac aaatgattta cgaagacggg ggcatttgtg tcgcgacaaa
      841 cgacataacc ctggatggtg actgtatgat ctccgaaatt cgatttaagg gtgtgaactt
      901 tcctgccaat ggtccagttt tccagaagag gactgtgaaa tgggagctat cccacgagaa
      961 attgtatgct cgtgatggac tgctgtactc tgatggtaac tacgctctgt cgcttgaagg
     1021 aggtggccat taccgatgtg acaataaaac tacttataaa gctaagaagg ttgtccagtt
     1081 gccagactat cactgggtga cccacagtat tgtgattaaa agccacgaca aagattacag
     1141 taatgttaat ctgcatgagc atgccgaagc acattctgag ctgccgaggc aggcccgaga
     1201 ccagttaacc gaggaacaaa tagctgagtt caaggaagcc ttttctttgt tcgataaaga
     1261 tggtgacggc accatcacca ctaaagaatt gggcactgtc atgaggagcc ttggtcaaaa
     1321 tccaactgag gctgaattgc aagatatgat taacgaggtc gacgctgatg gagatggtac
     1381 tatagatttt ccagaattct tgactatgat ggccaggaag atgaaggata ctgattcgga
     1441 agaggagata cgtgaggcct ttagagtatt cgacaaagat ggtaacggtt atattggtgc
     1501 cgctgaatta agacatgtta tgactaatct tggggaaaag ttgacggacg aggaagtcga
     1561 cgagatgatt agagtcgctg atattgacgg cgacggccaa gttaattatg aggaattcgt
     1621 gcagatgatg actacaaaat aggctagcgc agccaacgac gaaaactacg ctctagccgc
     1681 atagctcgag aagcttgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1741 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1801 ttttttgctg aaaggaggaa ctatatccgg atctggcgta atagcgaaga ggcccgcacc
     1861 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     1921 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1981 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2041 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2101 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2161 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2221 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2281 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2341 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2401 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2461 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2521 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2581 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2641 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2701 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2761 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2821 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2881 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2941 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3001 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3061 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3121 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3181 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3241 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3301 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3361 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3421 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3481 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3541 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3601 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3661 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3721 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3781 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3841 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3901 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3961 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4021 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4081 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4141 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4201 ataaccgtat taccgccttt gagtgagctg ataccg
//
