LOCUS       sequence-377044-        6541 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 6541)
  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 6541)
  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(+)-NEMOf
FEATURES             Location/Qualifiers
     source          1..6541
                     /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(2334..2351)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2340..2564
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2610..3038
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2697..2716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2907..2928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3047..3067)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3052..3381
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3232..3367
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3294..3313
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3402..4001
                     /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(3402..3421)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3898..3918
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4214..4335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4251..4270)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4305..4324
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4384..4400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4384..4400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4397..4419)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4408..4424
                     /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(4432..4438,4439..4456,4457..4462))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4477..4498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4615..4632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4786..5374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4866..4885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5545..6336,6337..6405))
                     /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     6168..6187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6406..6510)
                     /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 cccacgacga tctgacagaa gaacagatcg ccgaattcaa
     1501 ggaggagttt agcctgttcg acaaggacgg cgacggcacc attaccacaa aagagctggg
     1561 cacagtgttc 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 gcggaggcag
     1921 cagaagaaaa tggaataagg ccggccacgc cgtgagagcc attggcagac tttctagcat
     1981 ctacttcgcc gactggtgcg tgagcaagaa gacctgccct aacgacaaga ccatcgtgag
     2041 caccttcaag tgggccttca tcaccgacaa cggcaagaga tacagaagca ccgccagaac
     2101 cacctacacc ttcgccaaac ccatggccgc taactacctg aagaaccagc ccatgtacgt
     2161 gttcagaaag accgagctga agcacagcaa gaccgagctg aacttcaagg agtggcagaa
     2221 ggccttcacc gacgtgatgg gcatggacga gctgtacaag taagaattct gcagatatcc
     2281 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2341 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2401 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2461 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2521 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2581 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2641 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2701 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2761 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2821 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2881 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2941 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3001 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3061 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3121 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3181 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3241 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3301 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3361 ggaggcctag gcttttgcaa aaagctccca tcaagcttac catgaccgag tacaagccca
     3421 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     3481 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     3541 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3601 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     3661 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     3721 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     3781 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     3841 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     3901 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     3961 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     4021 agcgcccgac cgaaaggagc gcacgacccc atgcatcgaa atgaccgacc aagcgacgcc
     4081 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4141 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4201 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4261 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4321 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4381 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4441 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4501 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4561 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4621 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4681 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4741 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4801 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4861 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4921 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4981 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5041 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5101 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5161 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5221 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5281 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5341 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5401 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5461 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5521 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5581 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5641 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5701 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5761 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5821 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5881 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5941 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6001 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6061 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6121 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6181 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6241 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6301 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6361 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6421 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6481 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6541 c
//
