LOCUS       sequence-194122-        4520 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pTriFC_aidB. Expresses protein-NYFP and 2MS2BD-5' UTR mStrawberry 
            fusions in E. coli for Dual-fluorescence TriFC assay (ampR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4520)
  AUTHORS   Sowa SW, Gelderman G, Leistra AN, Buvanendiran A, Lipp S, Pitaktong 
            A, Vakulskas CA, Romeo T, Baldea M, Contreras LM
  TITLE     Integrative FourD omics approach profiles the target network of the 
            carbon storage regulatory system.
  JOURNAL   Nucleic Acids Res. 2017 Feb 28;45(4):1673-1686. doi: 
            10.1093/nar/gkx048.
  PUBMED    28126921
REFERENCE   2  (bases 1 to 4520)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2017-02-28- 28"; volume: "45"; issue: 
            "4"; pages: "1673-1686"
COMMENT     Sequence Label: pCML 961
FEATURES             Location/Qualifiers
     source          1..4520
                     /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"
     CDS             complement(1453..2160)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent 
                     protein"
                     /label=mStrawberry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILTPNFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKMRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVELYERAE
                     GRHSTGGMDELYK"
     primer_bind     2000..2018
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_RNA        complement(2373..2391)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(2412..2430)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     protein_bind    2533..2549
                     /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(2557..2563,2564..2581,2582..2587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2602..2623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2778..2882
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2883..2951,2952..3743)
                     /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(3101..3120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3914..4502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4403..4422
                     /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 aagggagcac tgtattcaca
     1441 gcgctcccgg ttctacttgt acagctcgtc catgccgccg gtggagtggc ggccctcggc
     1501 gcgttcgtac agttccacga tggtgtagtc ctcgttgtgg gaggtgatgt ccaacttgat
     1561 gccgacgatg taggcgccgg gcagctgcac gggcttcttg gccttgtagg tggtcttgac
     1621 ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc atcttgatct cgcccttcag
     1681 ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc cagcccatgg ttttcttctg
     1741 cattacgggg ccgtcggagg ggaagttggt gccgcgcagc ttcaccttgt agatgaactc
     1801 gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat
     1861 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc
     1921 ggggtgcttc acgtaggcct tggagccgta ggtgaagttg ggggttagga tgtcccaggc
     1981 gaagggcagg gggccaccct tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg
     2041 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggagc cctccatgcg
     2101 caccttgaag cgcatgaact ccttgatgat ggccatgtta ttctcctcgc ccttgctcac
     2161 acgtcttcgc atgccacgcg ttaccgcttc gcagagcgcg ccatcagaca ggtataagtt
     2221 gctgttattt aatggtatag gttgattaaa aacggtgtga gtttgccagt gcactgtgtc
     2281 tccctccatc aatggcagtc acactagtgt gcgcaaattt aaagcgctga tatcgatcgc
     2341 gcgcagatct aatgaacccg ggaatactgc agacatgggt gatcctcatg ttttctggag
     2401 tcgacctgca gacatgggtg atcctcatgt tttctaggca attaggtacc ttaggatcca
     2461 tatatagggc ccgggttata attacctcag gacgcgtggc gccctcgagc atatgctgtt
     2521 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2581 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2641 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2701 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgcgaccgg tcaggtggca
     2761 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2821 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2881 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2941 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3001 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3061 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3121 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3181 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3241 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3301 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3361 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3421 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3481 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3541 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3601 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3661 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3721 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3781 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3841 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3901 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3961 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4021 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4081 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4141 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4201 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4261 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4321 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4381 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4441 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4501 aaaacgccag caacgcggcc
//
