LOCUS       sequence-300770-        6695 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 6695)
  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 6695)
  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: CyPet-A206K-PRL2
FEATURES             Location/Qualifiers
     source          1..6695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             1415..1437
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1445..2158
                     /codon_start=1
                     /product="cyan fluorescent protein optimized for FRET with 
                     YPet (Nguyen and Daugherty, 2005)"
                     /label=CyPet
                     /translation="MSKGEELFGGIVPILVELEGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSVMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKARHNITDGSVQLADHYQQNTPIGDGPVILPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2678..2701
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2711..2734
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2744..2776
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2783..2800
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2853..2871)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2867..2914
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2951..3406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3038..3057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3248..3269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3432..3536
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3537..3605,3606..4397)
                     /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(3755..3774)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4568..5156
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5057..5076
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5310..5327
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5342..5481
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5407..5426)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5567..5589
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5583..5774)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6583..6695,1..970))
                     /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 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 cataggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttctgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     1441 acatatgtct aaaggtgaag aattattcgg cggtatcgtc ccaattttag ttgaattaga
     1501 gggtgatgtt aatggtcaca aattttctgt ctccggtgaa ggtgaaggtg atgctacgta
     1561 cggtaaattg accttaaaat ttatttgtac tactggtaaa ttgccagttc catggccaac
     1621 cttagtcact actctgactt ggggtgttca atgtttttct agatacccag atcatatgaa
     1681 acaacatgac tttttcaagt ctgtcatgcc agaaggttat gttcaagaaa gaactatttt
     1741 tttcaaagat gacggtaact acaagaccag agctgaagtc aagtttgaag gtgatacctt
     1801 agttaataga atcgaattaa aaggtattga ttttaaagaa gatggtaaca ttttaggtca
     1861 caaattggaa tacaactata tctctcacaa tgtttacatc accgctgaca aacaaaagaa
     1921 tggtatcaaa gctaacttca aagccagaca caacattacc gatggttctg ttcaattagc
     1981 tgaccattat caacaaaata ctccaattgg tgatggtcca gtcatcttgc cagacaacca
     2041 ttacttatcc actcaatcta aattatctaa agatccaaac gaaaagagag accacatggt
     2101 cttgctcgaa tttgttactg ctgctggtat tacccatggt atggatgaat tgtacaaagg
     2161 aagtaaccgt ccagcccctg tggagatctc ctatgagaac atgcgttttc tgataactca
     2221 caaccctacc aatgctactc tcaacaagtt cacagaggaa cttaagaagt atggagtgac
     2281 gactttggtt cgagtttgtg atgctacata tgataaagct ccagttgaaa aagaaggaat
     2341 ccacgttcta gattggccat ttgatgatgg agctccaccc cctaatcaga tagtagatga
     2401 ttggttaaac ctgttaaaaa ccaaatttcg tgaagagcca ggttgctgtg ttgcagtgca
     2461 ttgtgttgca ggattgggaa gggcacctgt gctggttgca cttgctttga ttgaatgtgg
     2521 aatgaagtac gaagatgcag ttcagtttat aagacaaaaa agaaggggag cgttcaattc
     2581 caaacagctg ctttatttgg agaaataccg acctaagatg cgattacgct tcagagatac
     2641 caatgggcat tgctgtgttc agctcgagag tagtggcctg gaagttctgt tccaggggcc
     2701 ctttactagt catcaccatc accatcacca tcactgagct agccagccag aactcgcccc
     2761 ggaagacccc gaggatgtcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     2821 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     2881 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     2941 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3001 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3061 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3121 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3181 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3241 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3301 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3361 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     3421 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3481 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3541 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3601 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3661 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3721 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3781 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3841 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3901 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3961 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4021 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4081 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4141 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4201 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4261 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4321 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4381 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4441 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4501 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4561 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4621 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4681 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4741 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4801 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4861 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4921 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4981 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5041 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5101 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5161 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5221 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5281 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5341 gcctgatgcg gtattttctc cttgcgcatc tgtgcggtat ttcacaccgc aatggtgcac
     5401 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     5461 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5521 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5581 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     5641 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     5701 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5761 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5821 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     5881 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     5941 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6001 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6061 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6121 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6181 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     6241 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     6301 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     6361 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     6421 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     6481 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     6541 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     6601 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6661 ttgcgtattg ggcgccaggg tggtttttct tttca
//
