LOCUS       sequence-377042-        6544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of calcium sensor with ultra-high dynamics and 
            sensitivity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6544)
  AUTHORS   Li J, Shang Z, Chen JH, Gu W, Yao L, Yang X, Sun X, Wang L, Wang T, 
            Liu S, Li J, Hou T, Xing D, Gill DL, Li J, Wang SQ, Hou L, Zhou Y, 
            Tang AH, Zhang X, Wang Y
  TITLE     Engineering of NEMO as calcium indicators with large dynamics and 
            high sensitivity.
  JOURNAL   Nat Methods. 2023 Jun;20(6):918-924. doi: 
            10.1038/s41592-023-01852-9.
  PUBMED    37081094
REFERENCE   2  (bases 1 to 6544)
  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"; journalName: 
            "Nat Methods"; date: "2023-06"; volume: "20"; issue: "6"; pages: 
            "918-924"
COMMENT     Sequence Label: pCDNA3.1(+)-NEMOm
FEATURES             Location/Qualifiers
     source          1..6544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1007..1024
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2337..2354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2343..2567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2613..3041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2700..2719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2910..2931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3050..3070)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3055..3384
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3235..3370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3297..3316
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3405..4004
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3405..3424)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3901..3921
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4217..4338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4254..4273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4308..4327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4387..4403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4387..4403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4400..4422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4411..4427
                     /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(4435..4441,4442..4459,4460..4465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4480..4501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4618..4635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4789..5377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4869..4888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5548..6339,6340..6408))
                     /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     6171..6190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6409..6513)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccgccacc atgctgcaga acgagcttgc
      961 tcttaagttg gctggacttg atattaacaa gactggagga ggttctcatc atcatcatca
     1021 tcatgtgagc aagggcgagg aggagaacat ggccagcctg cctgctacac atgaactgca
     1081 catttttgga agcatcaacg gcatcgactt cgacatggtg ggccagggca ctggaaatcc
     1141 taatgacgga tacgaggagc tgaatctgaa gagcaccatg ggcgacctgc agttcagccc
     1201 ctggatactg gtgcctcata ttggctatgg cttccaccaa tacctgccct accccgacgg
     1261 aatgagcccc tttcaagctg ccatggtgga tggcagcgga taccaagtgc atagaaccat
     1321 gcagttcgag gacggcgcca gccttacagt gaattataga tatacctacg agggcagcca
     1381 catcaagggc gaggcacaag tggaaggcac aggatttccc gctgatggcc ccgtgatgac
     1441 caatagcctg accgctgaag cccacgacca gctgacagaa gaacagatcg ccgaattcaa
     1501 ggaggccttt agcctgttcg acaaggacgg cgacggcacc attaccacaa aagagctggg
     1561 cacagtgatg agaagcctgg gccaaaatcc cacagaagct gagctgagag tgatgatcat
     1621 cgaggtggac gccgacggcg atggaacact ggattttcct gaatttctgg ccatgatggc
     1681 cagaaagatg aagtacagag acaccgagga ggagatcaga gaggccttcg gcgtgtttga
     1741 caaggacggc aatggataca ttggcgccgc tgaactgaga cacgtgatga ccaatctggg
     1801 cgagaagctg accgacgagg aggtgggaga actgatcaga gaggctgaca ttgacggcga
     1861 cggccaggtg aattatgagg agtttgtgca gatgatgacc gccaagggcg gcagcggagg
     1921 aagcagaaga aaatggaata aggccggcca cgccgtgaga gccattggca gactttctag
     1981 catctacttc gccgactggt gcgtgagcaa gaagacctgc cctaacgaca agaccatcgt
     2041 gagcaccttc aagtgggcct tcatcaccga caacggcaag agatacagaa gcaccgccag
     2101 aaccacctac accttcgcca aacccatggc cgctaactac ctgaagaacc agcccatgta
     2161 cgtgttcaga aagaccgagc tgaagcacag caagaccgag ctgaacttca aggagtggca
     2221 gaaggccttc accgacgtga tgggcatgga cgagctgtac aagtaagaat tctgcagata
     2281 tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2341 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2401 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2461 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2521 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2581 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2641 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2701 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2761 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2821 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2881 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2941 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3001 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3061 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3181 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3241 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3301 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3361 tttggaggcc taggcttttg caaaaagctc ccatcaagct taccatgacc gagtacaagc
     3421 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     3481 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     3541 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3601 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3661 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3721 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3781 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3841 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3901 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3961 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     4021 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc gaaatgaccg accaagcgac
     4081 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4141 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4201 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4261 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4321 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4381 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4441 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5341 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5401 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5521 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5581 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5641 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5701 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5761 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5821 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5881 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5941 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6001 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6061 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6121 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6181 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6241 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6301 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6361 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6421 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6481 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6541 cgtc
//
