LOCUS       sequence-377045-        6478 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 6478)
  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 6478)
  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(+)-NEMOc
FEATURES             Location/Qualifiers
     source          1..6478
                     /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             944..961
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2271..2288)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2277..2501
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2547..2975
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2634..2653)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2844..2865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2984..3004)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2989..3318
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3169..3304
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3231..3250
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3339..3938
                     /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(3339..3358)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3835..3855
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4151..4272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4188..4207)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4242..4261
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4321..4337)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4321..4337)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4334..4356)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4345..4361
                     /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(4369..4375,4376..4393,4394..4399))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4414..4435
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4552..4569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4723..5311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4803..4822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5482..6273,6274..6342))
                     /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     6105..6124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6343..6447)
                     /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 atgcatcatc atcatcatca
      961 tgtgagcaag ggcgaggagg agaacatggc cagcctgcct gctacacatg aactgcacat
     1021 ttttggaagc atcaacggca tcgacttcga catggtgggc cagggcactg gaaatcctaa
     1081 tgacggatac gaggagctga atctgaagag caccatgggc gacctgcagt tcagcccctg
     1141 gatactggtg cctcatattg gctatggctt ccaccaatac ctgccctacc ccgacggaat
     1201 gagccccttt caagctgcca tggtggatgg cagcggatac caagtgcata gaaccatgca
     1261 gttcgaggac ggcgccagcc ttacagtgaa ttatagatat acctacgagg gcagccacat
     1321 caagggcgag gcacaagtgg aaggcacagg atttcccgct gatggccccg tgatgaccaa
     1381 tagcctgacc gctgaagccc acgaccagct gacagaagaa cagatcgccg aattcaagga
     1441 ggcctttagc ctgttcgaca aggacggcga cggcaccatt accacaaaag agctgggcac
     1501 agtgatgaga agcctgggcc aaaatcccac agaagctgag ctgagagtga tgatcatcga
     1561 ggtggacgcc gacggcgatg gaacactgga ttttcctgaa tttctggcca tgatggccag
     1621 aaagatgaag tacagagaca ccgaggagga gatcagagag gccttcggcg tgtttgacaa
     1681 ggacggcaat ggatacattg gcgccgctga actgagacac gtgatgacca atctgggcga
     1741 gaagctgacc gacgaggagg tgggagaact gatcagagag gctgacattg acggcgacgg
     1801 ccaggtgaat tatgaggagt ttgtgcagat gatgaccgcc aagggcggcg gaggcagcag
     1861 aagaaaatgg aataaggccg gccacgccgt gagagccatt ggcagacttt ctagcatcta
     1921 cttcgccgac tggtgcgtga gcaagaagac ctgccctaac gacaagacca tcgtgagcac
     1981 cttcaagtgg gccttcatca ccgacaacgg caagagatac agaagcaccg ccagaaccac
     2041 ctacaccttc gccaaaccca tggccgctaa ctacctgaag aaccagccca tgtacgtgtt
     2101 cagaaagacc gagctgaagc acagcaagac cgagctgaac ttcaaggagt ggcagaaggc
     2161 cttcaccgac gtgatgggca tggacgagct gtacaagtaa gaattctgca gatatccagc
     2221 acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2281 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2341 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2401 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2461 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2521 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2581 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2641 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2701 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2761 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2821 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2881 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2941 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3001 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3061 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3121 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3181 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3241 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3301 ggcctaggct tttgcaaaaa gctcccatca agcttaccat gaccgagtac aagcccacgg
     3361 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     3421 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     3481 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     3541 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     3601 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     3661 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     3721 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     3781 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     3841 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     3901 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc
     3961 gcccgaccga aaggagcgca cgaccccatg catcgaaatg accgaccaag cgacgcccaa
     4021 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4081 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4141 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4201 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4261 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4321 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4381 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4441 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4501 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4561 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4621 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4681 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4741 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4801 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4861 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4921 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4981 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5041 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5101 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5161 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5221 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5281 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5341 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5401 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6361 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6421 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
