LOCUS       sequence-194123-        3999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCsrA-NYFP + CsrB-2MS2BD. Expresses protein-NYFP and RNA-2MS2BD 
            fusions in E. coli for TriFC assay (ampR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3999)
  AUTHORS   Gelderman G, Sivakumar A, Lipp S, Contreras L
  TITLE     Adaptation of Tri-molecular fluorescence complementation allows 
            assaying of regulatory Csr RNA-protein interactions in bacteria.
  JOURNAL   Biotechnol Bioeng. 2015 Feb;112(2):365-75. doi: 10.1002/bit.25351. 
            Epub 2014 Sep 26.
  PUBMED    25080893
REFERENCE   2  (bases 1 to 3999)
  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"; doi: "10.1002/bit.25351";
            journalName: "Biotechnol Bioeng"; date: "2015-02"; volume: "112"; 
            issue: "2"; pages: "365-75"
COMMENT     Sequence Label: pCML 314
FEATURES             Location/Qualifiers
     source          1..3999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     136..153
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    270..291
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(306..311,312..329,330..336)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    344..360
                     /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     368..384
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     368..384
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(653..655,656..1114)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     primer_bind     complement(695..716)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(956..975)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1115..1138
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1176..1195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1295..1317
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1355..1373)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(1526..1544)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(1565..1583)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     protein_bind    2012..2028
                     /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(2036..2042,2043..2060,2061..2066))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2081..2102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2257..2361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2362..2430,2431..3222)
                     /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(2580..2599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3393..3981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3882..3901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
       61 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      121 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      181 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      241 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      301 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      361 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgatgctga ttctgactcg
      421 tcgagttggt gagaccctca tgattgggga tgaggtcacc gtgacagttt taggggtaaa
      481 gggcaaccag gtacgtattg gcgtaaatgc cccgaaggaa gtttctgttc accgtgaaga
      541 gatctaccag cgtatccagg ctgaaaaatc ccagcagtcc agttacgagc tcgctagcga
      601 attcggcggc ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgtgagcaa
      661 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
      721 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
      781 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
      841 cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
      901 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
      961 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1021 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1081 caactacaac agccacaacg tctatatcat ggccgattat aaagacgatg acgataaata
     1141 aatcgatata actaagtaat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     1201 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     1261 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     1321 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     1381 gttaatgtca tgataataat ggtttcttag acgtcaataa aaaaagggag cactgtattc
     1441 acagcgctcc cggttactag tgtgcgcaaa tttaaagcgc tgatatcgat cgcgcgcaga
     1501 tctaatgaac ccgggaatac tgcagacatg ggtgatcctc atgttttctg gagtcgacct
     1561 gcagacatgg gtgatcctca tgttttctag gcaattaggt accttaggat ccatatatag
     1621 ggcccgggtt ataattacct caggacgcgt ggcgcccgtt tcgcagcatt ccagctactt
     1681 ttgttgctcc ctgctcatcc ttgacaactt ttcctctggc cttgcggcca atcgttcatc
     1741 ctgaactatt gcttcctgct cacaccaccc cgatgtgata cttcatcctg aagtgtccct
     1801 ggccttcctg acccaccgaa tcatcctgac cggttctcat tctccatcct ggaggtgtcc
     1861 tttaacgcgt cctgcgtcat cctcttcgct tcatccagaa gcctttccct gaaacaccat
     1921 cctggtgtgt cctgcagaag tgtcatcatc ctgatgttca cttcgttgtc tgactccctg
     1981 tcgacggtca tatgctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2041 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2101 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2161 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2221 cgcgaccggt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2281 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2341 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2401 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2461 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2521 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2581 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2641 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2701 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2761 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2821 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2881 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2941 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3001 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3061 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3121 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3181 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3241 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3301 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3361 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3421 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3481 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3541 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3601 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3661 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3721 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3781 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3841 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3901 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3961 ggggggcgga gcctatggaa aaacgccagc aacgcggcc
//
