LOCUS       sequence-377043-        6580 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 6580)
  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 6580)
  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(+)-NEMOs
FEATURES             Location/Qualifiers
     source          1..6580
                     /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             1034..1051
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2373..2390)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2379..2603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2649..3077
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2736..2755)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2946..2967
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3086..3106)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3091..3420
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3271..3406
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3333..3352
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3441..4040
                     /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(3441..3460)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3937..3957
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4253..4374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4290..4309)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4344..4363
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4423..4439)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4423..4439)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4436..4458)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4447..4463
                     /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(4471..4477,4478..4495,4496..4501))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4516..4537
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4654..4671)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4825..5413)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4905..4924)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5584..6375,6376..6444))
                     /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     6207..6226
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6445..6549)
                     /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 gactggaggg atcactctcg gcatggacga
     1021 gctttacaag atgcatcatc atcatcatca tgtgagcaag ggcgaggagg agaacatggc
     1081 cagcctgcct gctacacatg aactgcacat ttttggaagc atcaacggca tcgacttcga
     1141 catggtgggc cagggcactg gaaatcctaa tgacggatac gaggagctga atctgaagag
     1201 caccatgggc gacctgcagt tcagcccctg gatactggtg cctcatattg gctatggctt
     1261 ccaccaatac ctgccctacc ccgacggaat gagccccttt caagctgcca tggtggatgg
     1321 cagcggatac caagtgcata gaaccatgca gttcgaggac ggcgccagcc ttacagtgaa
     1381 ttatagatat acctacgagg gcagccacat caagggcgag gcacaagtgg aaggcacagg
     1441 atttcccgct gatggccccg tgatgaccaa tagcctgacc gctgaagccc acgaccagct
     1501 gacagaagaa cagatcgccg aattcaagga ggcctttagc ctgttcgaca aggacggcga
     1561 cggcaccatt accacaaaag agctgggcac agtgatgaga agcctgggcc aaaatcccac
     1621 agaagctgag ctgagagtga tgatcatcga ggtggacgcc gacggcgatg gaacactgga
     1681 ttttcctgaa tttctggcca tgatggccag aaagatgaag tacagagaca ccgaggagga
     1741 gatcagagag gcctttggcg tgtttgacaa ggacggcaat ggatacattg gcgccgctga
     1801 actgagacac gtgatgacca atctgggcga gaagctgacc gacgaggagg tgggagaact
     1861 gatcagagag gctgacattg acggcgacgg ccaggtgaat tatgaggagt ttgtgcagat
     1921 gatgaccgcc aagggcggca gcggaggagg aagcagcagc agaagaaaat ggaataaggc
     1981 cggccacgcc gtgagagcca ttggcagact ttctagcatc tacttcgccg actggtgcgt
     2041 gagcaagaag acctgcccta acgacaagac catcgtgagc accttcaagt gggccttcat
     2101 caccgacaac ggcaagagat acagaagcac cgccagaacc acctacacct tcgccaaacc
     2161 catggccgct aactacctga agaaccagcc catgtacgtg ttcagaaaga ccgagctgaa
     2221 gcacagcaag accgagctga acttcaagga gtggcagaag gccttcaccg acgtgatggg
     2281 catggacgag ctgtacaagt aagaattctg cagatatcca gcacagtggc ggccgctcga
     2341 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2401 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2461 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2521 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2581 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2641 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2701 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2761 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2821 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2881 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2941 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3001 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3061 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3181 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3241 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3301 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3361 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3421 aagctcccat caagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3481 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3541 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3601 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     3661 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     3721 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     3781 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     3841 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     3901 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     3961 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4021 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     4081 cacgacccca tgcatcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4141 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4201 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4261 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4321 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4381 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4441 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4501 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4561 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4621 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4681 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4741 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4801 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4861 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4921 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4981 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5041 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5101 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5161 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5221 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5281 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5341 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5401 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5461 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5521 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5581 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5641 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5701 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5761 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5821 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5881 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5941 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6001 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6061 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6121 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6181 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6241 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6301 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6361 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6421 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6481 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6541 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
