LOCUS       sequence-364342-        6992 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For expression of a fluorescent sensor for neuromedin B in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6992)
  AUTHORS   Ino D, Tanaka Y, Hibino H, Nishiyama M
  TITLE     A fluorescent sensor for real-time measurement of extracellular 
            oxytocin dynamics in the brain.
  JOURNAL   Nat Methods. 2022 Sep 22. pii: 10.1038/s41592-022-01597-x. doi: 
            10.1038/s41592-022-01597-x.
  PUBMED    36138174
REFERENCE   2  (bases 1 to 6992)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2022 Sep 22. pii: 10.1038/s41592-022-01597-x. doi: 
            10.1038/s41592-022-01597-x."
COMMENT     Sequence Label: pCIS.MTRIA-NMB
FEATURES             Location/Qualifiers
     source          1..6992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..583
                     /label=chicken beta-actin promoter
     intron          584..1600
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1524..1546
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1608..1627
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1656..1672
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1657..1673
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2608..2629
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2735..2756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3519..3535)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3519..3535)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3624..3643)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3689..3744
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3743..3762)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(4099..4117)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4138..4154)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4138..4154)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4151..4173)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4162..4178
                     /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(4186..4192,4193..4210,4211..4216))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4231..4252
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4369..4386)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4540..5128)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4620..4639)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5299..6090,6091..6159))
                     /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     5922..5941
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6160..6264)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6291..6746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6378..6397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6588..6609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6872..6894
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6886..6903
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6887..6903
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6910..6929
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6910..6928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
      481 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      541 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
      601 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
      661 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg
      721 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg
      781 ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg
      841 gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg
      901 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
      961 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     1021 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
     1081 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
     1141 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
     1201 ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     1261 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     1321 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     1381 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     1441 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     1501 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     1561 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     1621 tattgtgctg tctcatcatt ttggcaaagg attcctcgag gtcgacggta tcgataagct
     1681 tgatatcgaa ttcaccatgg atcatacttt ctccgaggat accgcagtct acgagaactt
     1741 cacagaggac tggacgtcag atggagggga aaccgtgcat ttcatcattc aatgcgttat
     1801 cccgtcggtc tacattctta tcatcacggt tggtttactg ggaaacatca ctctcgtgaa
     1861 aatatttatt actacaagcg caatgcgaag tgtgccaaac attttcatct ccagcttggc
     1921 tgtgggggat cttttgcttt tggtcacttg tgtgccagtg gacgcgttca ggtatttcta
     1981 cgaggagtgg atcttcggaa cggtggcttg taagatgatc ccggtgatcc agctcacctc
     2041 ggtcggagtc tccgtgttca ctctcacggc actgagcgcg gacagacaca aagctattgt
     2101 gaatcccatg gacatccagt cgtccagcgc ggtgttctgg acctgcttga aggctatcac
     2161 tatatgggtg gtttcggtgc tgctggccat ccctgaggcg gttttctcac aggtggttca
     2221 tatgccagac aagaacacga cgttcaccgc ctgcgtgcca tatccgctgt ccaacgagat
     2281 gcatcctaag attcactcca ttatgatatt cctggtctac ttcctcatcc ctctgagcat
     2341 tatatctgtt tactactacc acatctgcta caaaatatgg cgaaatttca aagggaaatt
     2401 aaacgtctat atcaaggccg acaagcagaa gaacggcatc aaggcgaact tccacatccg
     2461 ccacaacatc gaggacggcg gcgtgcagct cgcctaccac taccagcaga acacccccat
     2521 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcgtgcaga ccaaactttc
     2581 gaaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2641 gatcactctc ggcatggacg agctgtacaa gggcggtacc ggagggatga tggtgagaaa
     2701 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2761 cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac
     2821 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2881 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2941 cttcaagtcc gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga
     3001 cggccactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3061 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3121 caactcagta atctccaagg caaagatacg cacagtgaaa atggttctgg tattcgtggg
     3181 cctctttgcc ctgtgctggt tccccaacca cgttctctac atgtaccgct cattcaacta
     3241 tcggcagatc gactcctcgc tgtcacacct catcatcacg cttgtggcac gcgtgctgag
     3301 cttctccagc tcctgcgtca acccgtttgc cttgtatctg ctgagcgaga gcttccgacg
     3361 ccacttcaac aaccagctgc tgtgtagaca acgcaaatac caagagcgca acactagcta
     3421 cctccagagc acctctgcca tccgcatgac ctccatccgg aagagcacac ctgccgtcgc
     3481 caacggccat ggccaaaaac aaggagtctg catttaggga tccactagtt ctagagcggc
     3541 cgcttcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     3601 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     3661 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     3721 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     3781 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     3841 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     3901 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     3961 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     4021 ctctcctgac tactcccagt catagctgtc cctcttctct tatgaagatc cctcgacctg
     4081 gagctccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt aatcatggtc
     4141 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4201 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4261 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4321 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4381 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4441 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4501 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4561 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4621 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4681 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4741 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4801 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4861 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4921 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4981 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5041 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5101 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5161 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5221 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5281 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5341 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5401 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5461 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5521 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5581 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5641 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5701 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5761 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5821 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5881 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5941 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6001 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6061 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6121 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6181 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6241 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     6301 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     6361 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     6421 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     6481 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     6541 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     6601 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     6661 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     6721 taacaaaata ttaacgctta caatttccat tcgccattca ggctgcgcaa ctgttgggaa
     6781 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6841 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     6901 agtgaattgt aatacgactc actatagggc gaattgggta cctagtaatc aattacgggg
     6961 tcattagttc atagcccata tatggagttc cg
//
