LOCUS       sequence-364867-        7236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of a fluorescent sensor for neurokinin B in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7236)
  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 7236)
  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"; 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-NKB
FEATURES             Location/Qualifiers
     source          1..7236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..581
                     /label=chicken beta-actin promoter
     intron          582..1598
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1522..1544
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1606..1625
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1654..1670
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1655..1671
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2299..2310)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2708..2729
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2835..2856)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3763..3779)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3763..3779)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3868..3887)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3933..3988
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3987..4006)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(4343..4361)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4382..4398)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4382..4398)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4395..4417)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4406..4422
                     /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(4430..4436,4437..4454,4455..4460))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4475..4496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4613..4630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4784..5372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4864..4883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5543..6334,6335..6403))
                     /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     6166..6185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6404..6508)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6535..6990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6622..6641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6832..6853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7116..7138
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7130..7147
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7131..7147
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7154..7173
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7154..7172
                     /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 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
      481 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      541 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      601 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      661 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      721 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      781 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      841 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      901 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      961 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1021 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1081 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1141 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1201 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1261 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1321 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1381 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1441 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1501 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1561 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1621 ttgtgctgtc tcatcatttt ggcaaaggat tcctcgaggt cgacggtatc gataagcttg
     1681 atatcgaatt caccatggcc tcggttccca ccggcgaaaa ctggacggac gggacggcgg
     1741 gcgtggggtc ccatacaggg aatctgagcg ctgcgctggg gatcactgag tggcttgcgc
     1801 tccaggctgg caacttctcc tccgcgctgg gcttgccagt gacatcccag gcaccttccc
     1861 aagtccgggc caacctgacg aaccagttcg tgcagccctc ctggcgaatc gcgctctggt
     1921 cgctggccta tggcttagtg gtggcggtgg cagtcttcgg aaacctcatc gttatttgga
     1981 tcatcttggc ccacaagcgc atgaggaccg tcaccaacta tttcctggta aacctggctt
     2041 tctccgacgc ctccgtggct gccttcaaca ccttggtcaa tttcatctat ggtgttcaca
     2101 gcgagtggta ctttggcgcc aactactgcc gcttccagaa cttctttccc atcacagcgg
     2161 tgtttgccag catctactct atgacagcca ttgcagtgga caggtatatg gccattatcg
     2221 atcctttgaa acccagacta tctgccacag ccactaagat tgtcatcgga agtatttgga
     2281 ttttggcatt tctacttgcc ttccctcaat gtctttattc caaaataaaa gtcatgccag
     2341 gccgtaccct ttgctatgtg cagtggccag aaggtcccaa gcaacatttc acgtaccaca
     2401 tcattgttat catcctggtg tactgtttcc cattgctcat catgggtgtc acctacacca
     2461 tcgtttgcta caaaatatgg cgaaatttca aagggaaatt aaacgtctat atcaaggccg
     2521 acaagcagaa gaacggcatc aaggcgaact tccacatccg ccacaacatc gaggacggcg
     2581 gcgtgcagct cgcctaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2641 tgcccgacaa ccactacctg agcgtgcaga ccaaactttc gaaagacccc aacgagaagc
     2701 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2761 agctgtacaa gggcggtacc ggagggatga tggtgagaaa gggcgaggag ctgttcaccg
     2821 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2881 ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2941 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3001 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3061 aaggctacat ccaggagcgc accatcttct tcaaggacga cggccactac aagacccgcg
     3121 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     3181 tcaaggagga cggcaacatc ctggggcaca agctggagta caactcagta atctccaagg
     3241 caaagatacg cacagtgaaa atgatgatta ttgtggtggt gacatttgcc atctgctggc
     3301 taccctacca tgtgtatttc attctcactg cgatctacca acagttaaac aggtggaaat
     3361 acatccagca ggtctacctg gctagcttct ggctggccat gagctcaacc atgtacaacc
     3421 ccatcatcta ctgctgtttg aacaaaagat ttcgtgcagg cttcaagaga gcatttcgct
     3481 ggtgtccttt catccaagtc tccagctacg atgagctgga gctcaagacc accaggtttc
     3541 atcccacacg gcagagcagc ctgtacaccg tgagcaggat ggagtcagtg accgtgctgt
     3601 atgaccccag tgaaggggac ccggccaagt ccagccgcaa gaagagggcg gtgcccagag
     3661 accccagtgc caatggctgc tcccacagag agttcaaatc tgcctccacc acatcaagtt
     3721 tcataagctc gccctatacc tctgtggatg aatattccta aggatccact agttctagag
     3781 cggccgcttc actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     3841 tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat
     3901 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     3961 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     4021 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     4081 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     4141 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     4201 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     4261 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgaa gatccctcga
     4321 cctggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
     4381 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4441 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4501 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4561 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4621 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4681 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4741 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4801 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4861 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4921 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4981 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5041 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5101 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5161 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5221 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5281 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5341 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5401 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5461 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5521 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5581 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5641 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5701 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5761 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5821 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5881 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5941 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6001 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6061 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6121 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6181 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6241 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6301 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6361 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6421 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6481 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
     6541 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     6601 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     6661 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     6721 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     6781 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     6841 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     6901 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6961 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg
     7021 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     7081 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     7141 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtacctagt aatcaattac
     7201 ggggtcatta gttcatagcc catatatgga gttccg
//
