LOCUS       sequence-377046-        6553 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 6553)
  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 6553)
  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(+)-NEMOb
FEATURES             Location/Qualifiers
     source          1..6553
                     /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(2346..2363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2352..2576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2622..3050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2709..2728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2919..2940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3059..3079)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3064..3393
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3244..3379
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3306..3325
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3414..4013
                     /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(3414..3433)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3910..3930
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4226..4347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4263..4282)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4317..4336
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4396..4412)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4396..4412)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4409..4431)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4420..4436
                     /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(4444..4450,4451..4468,4469..4474))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4489..4510
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4627..4644)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4798..5386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4878..4897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5557..6348,6349..6417))
                     /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     6180..6199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6418..6522)
                     /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 aggaagcagc agcagaagaa aatggaataa ggccggccac gccgtgagag ccattggcag
     1981 actttctagc atctacttcg ccgactggtg cgtgagcaag aagacctgcc ctaacgacaa
     2041 gaccatcgtg agcaccttca agtgggcctt catcaccgac aacggcaaga gatacagaag
     2101 caccgccaga accacctaca ccttcgccaa acccatggcc gctaactacc tgaagaacca
     2161 gcccatgtac gtgttcagaa agaccgagct gaagcacagc aagaccgagc tgaacttcaa
     2221 ggagtggcag aaggccttca ccgacgtgat gggcatggac gagctgtaca agtaagaatt
     2281 ctgcagatat ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg
     2341 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2401 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2461 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2521 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2581 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2641 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2701 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2761 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2821 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2881 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2941 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3001 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3061 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3121 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3181 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3241 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3301 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3361 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc catcaagctt accatgaccg
     3421 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     3481 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     3541 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3601 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3661 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3721 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3781 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3841 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3901 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3961 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     4021 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg aaatgaccga
     4081 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4141 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4201 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4261 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4321 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4381 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4441 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4501 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4561 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4621 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4681 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4741 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4801 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4861 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4921 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4981 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5041 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5101 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5161 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5221 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5281 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5341 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5401 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5461 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5521 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5581 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5641 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5701 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5761 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5821 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5881 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5941 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6001 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6061 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6121 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6181 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6241 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6301 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6361 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6481 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6541 gccacctgac gtc
//
