LOCUS       sequence-300774-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the FRET-based binding assay between human CNNM3 CBS domain and 
            PRL2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  AUTHORS   Cai F, Huang Y, Wang M, Sun M, Zhao Y, Hattori M
  TITLE     A FRET-based screening method to detect potential inhibitors of the 
            binding of CNNM3 to PRL2.
  JOURNAL   Sci Rep. 2020 Jul 30;10(1):12879. doi: 10.1038/s41598-020-69818-x.
  PUBMED    32733084
REFERENCE   2  (bases 1 to 6662)
  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: "Sci Rep."; 
            date: "2020-07-30"; volume: "10(1):12879. doi"; pages: " 
            10.1038/s41598-020-69818-x"
COMMENT     Sequence Label: CNNM3-CBS-YPet
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     871..890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /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             1032..1054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1533..2246
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLE
                     FLTAAGITEGMNELYK"
     CDS             2262..2285
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2295..2318
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2328..2360
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2367..2384
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2437..2455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2451..2498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2535..2990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2622..2641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2832..2853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3016..3120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3121..3189,3190..3981)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3339..3358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4152..4740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4641..4660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4894..4911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4926..5065
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4991..5010)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5151..5173
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5167..5358)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6167..6662,1..587))
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSY
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat aggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttctgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
      721 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
      781 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
      841 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      901 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      961 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgtacagc gatctcagca
     1081 agggcgtgct gagatgcagg acagtggagg acgtgctcac ccccctggag gactgtttca
     1141 tgctggacgc cagcaccgtg ctcgactttg gcgtgctcgc ctccatcatg cagtccggcc
     1201 acacccgtat tcctgtgtat gaggaggaga ggtccaatat cgtggacatg ctctacctga
     1261 aggacctggc cttcgtggac cctgaggact gcacacctct gagcaccatc accaggttct
     1321 acaaccaccc cctccacttc gtgttcaatg acaccaaact ggacgccgtg ctggaggagt
     1381 tcaagagggg caagagccac ctggccatcg tgcagaaggt caacaatgag ggcgagggag
     1441 accccttcta cgaggtgctg ggcctggtga ccctggagga cgtcatcgag gagatcatca
     1501 ggtccgagat cctggacgag ctcgagggaa gtatgtctaa aggtgaagaa ttattcactg
     1561 gtgttgtccc aattttggtt gaattagatg gtgatgttaa tggtcacaaa ttttctgtct
     1621 ccggtgaagg tgaaggtgat gctacgtacg gtaaattgac cttaaaatta ctctgtacta
     1681 ctggtaaatt gccagttcca tggccaacct tagtcactac tttaggttat ggtgttcaat
     1741 gttttgctag atacccagat catatgaaac aacatgactt tttcaagtct gccatgccag
     1801 aaggttatgt tcaagaaaga actatttttt tcaaagatga cggtaactac aagaccagag
     1861 ctgaagtcaa gtttgaaggt gataccttag ttaatagaat cgaattaaaa ggtattgatt
     1921 ttaaagaaga tggtaacatt ttaggtcaca aattggaata caactataac tctcacaatg
     1981 tttacatcac tgctgacaaa caaaagaatg gtatcaaagc taacttcaaa attagacaca
     2041 acattgaaga tggtggtgtt caattagctg accattatca acaaaatact ccaattggtg
     2101 atggtccagt cttgttacca gacaaccatt acttatccta tcaatctgcc ttattcaaag
     2161 atccaaacga aaagagagac cacatggtct tgttagaatt tttgactgct gctggtatta
     2221 ccgagggtat gaatgaattg tacaaactcg agagtagtgg cctggaagtt ctgttccagg
     2281 ggccctttac tagtcatcac catcaccatc accatcactg agctagccag ccagaactcg
     2341 ccccggaaga ccccgaggat gtcgagcacc accaccacca ccactgagat ccggctgcta
     2401 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2461 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2521 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2581 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2641 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2701 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2761 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2821 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2881 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2941 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3061 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3121 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3241 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3301 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3361 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3421 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3481 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3661 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3721 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3781 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3961 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4021 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4261 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4801 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4861 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4921 gagcgcctga tgcggtattt tctccttgcg catctgtgcg gtatttcaca ccgcaatggt
     4981 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5041 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5101 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5161 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5221 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5281 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5341 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5401 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5461 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5521 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5581 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5641 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5701 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5761 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5821 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     5881 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     5941 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     6001 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     6061 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     6121 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     6181 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6241 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     6301 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     6361 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6421 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     6481 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6541 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6601 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6661 gc
//
