LOCUS       sequence-364431-        7059 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For expression of a fluorescent sensor for CCK in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7059)
  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 7059)
  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-CCK
FEATURES             Location/Qualifiers
     source          1..7059
                     /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"
     primer_bind     2687..2708
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2814..2835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3691..3710)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3756..3811
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3810..3829)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(4166..4184)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4205..4221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4205..4221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4229..4245
                     /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(4253..4259,4260..4277,4278..4283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4298..4319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4436..4453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4607..5195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4687..4706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5366..6157,6158..6226))
                     /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     5989..6008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6227..6331)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6358..6813
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6445..6464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6655..6676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6939..6961
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6953..6970
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6954..6970
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6977..6996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6977..6995
                     /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 caccatggat actttgagaa acgagacagc atcaaccatc gagtgcgtcg
     1741 ccggcgcgct gatggagcag cttcagagca gagcggctta caacgggagc ctggagactg
     1801 agaacatctc ctgcgtgaac ccgtccacgc ttccaacgcc gcagcgcgtg agacacagag
     1861 aagaggacca cacgctccgg atcctgcttt actcgctcat cttcttcctg agtgtctttg
     1921 gaaacctgct catcattgtg gtcctcactg tcaacaagcg aatgcgcacc gtcactaaca
     1981 ccttcctgct gtctctcgcc gtgagcgacc tgatgatggc catcttctgc atgcccttca
     2041 ccctcatccc caacatcctc aaggacttca tatttggagc agccatgtgc aagatcgtgt
     2101 cctacctcat gggcatatca gtgagcatct ccacgtttag cctggttgcc atagcaatcg
     2161 agcgctacag tgccatttgc aaccccctga agtcacgggt gtggcaaacc cgttcccatg
     2221 cctaccgcgt gattgctgct acatgggtgc tggccttcat catcatggtc ccctacccaa
     2281 ttatcagtca cctggagcct tttcagggtt ctgccaacac cacagcgcac aagtgtcgcc
     2341 acaagtggcc catccaaact gctgagcaga cctggtatgt aatgcttctg ctaatattgt
     2401 ttgccattcc tggagtggtt atgatcattg cctacggact gatctgctac aaaatatggc
     2461 gaaatttcaa agggaaatta aacgtctata tcaaggccga caagcagaag aacggcatca
     2521 aggcgaactt ccacatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact
     2581 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2641 gcgtgcagac caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2701 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
     2761 gagggatgat ggtgagaaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2821 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg
     2881 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2941 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3001 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
     3061 ccatcttctt caaggacgac ggccactaca agacccgcgc cgaggtgaag ttcgagggcg
     3121 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3181 tggggcacaa gctggagtac aactcagtaa tctccaaggc aaagatacgc acagtgaaaa
     3241 tgcttgtggt catcgtcgtc cttttctttc tctgttggat gcctctgtac tgtgtcaaca
     3301 cctggagggc ttttgatgac agctccgcca cccgtgctct ctccggagca cctattgctt
     3361 ttatccactt gctgagctac acctccgcct gtgtcaaccc catcatctac tgcttcatga
     3421 acactcgctt ccgcaaagcc ctgctctcca ccttctcgtg gtgcggcgct ccctgtcgtc
     3481 actgctgccg ccgtcgtggg ctacgcgaca tcgaggaaga cgttatggca acgggggcct
     3541 ccatgtcaaa gttcagttac actacagtca gcaccatggg aaactgctga actagttcta
     3601 gagcggccgc ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     3661 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     3721 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     3781 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     3841 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     3901 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3961 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     4021 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     4081 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct
     4141 cgacctggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     4201 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4261 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4321 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4381 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5041 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5221 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5341 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5401 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5461 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5521 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5581 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5641 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5701 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5761 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5821 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5881 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5941 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6001 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6061 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6121 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6181 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6241 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6301 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6361 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6421 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6481 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6541 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     6601 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     6661 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6721 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6781 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
     6841 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     6901 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     6961 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtacct agtaatcaat
     7021 tacggggtca ttagttcata gcccatatat ggagttccg
//
