LOCUS       sequence-364868-        6999 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For expression of a fluorescent sensor for enkephalin in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6999)
  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 6999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-ENK
FEATURES             Location/Qualifiers
     source          1..6999
                     /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     2627..2648
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2754..2775)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3526..3542)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3526..3542)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3631..3650)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3696..3751
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3750..3769)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(4106..4124)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4145..4161)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4145..4161)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4158..4180)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4169..4185
                     /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(4193..4199,4200..4217,4218..4223))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4238..4259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4376..4393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4547..5135)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4627..4646)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5306..6097,6098..6166))
                     /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     5929..5948
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6167..6271)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6298..6753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6385..6404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6595..6616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6879..6901
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6893..6910
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6894..6910
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6917..6936
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6917..6935
                     /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 gggggsgcgc 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 tcctcgagac catggagccg tccgtcattc
     1681 ccggtgctga catacccgac ctttactcca ttaacccgtt taatgtcact tttcccgacg
     1741 acgttttgag tttcgttcct gatgggagga actacaccga acctaacccg gtaaagagcc
     1801 ccgcgggaat catcatcgcc atttccatca ccgctcttta ctccgttatc tgcgttgtgg
     1861 gactcttagg aaacatccta gtgatgtacg gggtggtcag atacaccaag ctgaaaactg
     1921 ccaccaacat ctacatcttc aatttggccc tggcagatgc actggctact agtacacttc
     1981 cattccagag caccaagtat ttaatgaaca cctggccctt cggtgagctc ctttgcaaag
     2041 tggtcatagc catagactac tacaatatgt ttaccagtat cttcactctc actatgatga
     2101 gcgtggaccg ttacattgct gtgtgccacc ctgtgagagc actggagttt cgtaccccga
     2161 tcaaggccaa aataatcaat gtgtgcatct ggatcctctc ctccgctgtt ggagtgccaa
     2221 tcatgattat ggcagtcact agagtgacaa atcagaatac aacagtctgc atgctgaagt
     2281 tccctgatcc agactggtac tgggacacag tcacaaagat ctgtgtgttt atctttgcat
     2341 ttgtggtacc agtcctagtc ataacaatct gctatggtct gatgtgctac aaaatatggc
     2401 gaaatttcaa agggaaatta aacgtctata tcaaggccga caagcagaag aacggcatca
     2461 aggcgaactt ccacatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact
     2521 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2581 gcgtgcagac caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2641 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
     2701 gagggatgat ggtgagaaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2761 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg
     2821 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2881 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2941 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
     3001 ccatcttctt caaggacgac ggccactaca agacccgcgc cgaggtgaag ttcgagggcg
     3061 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3121 tggggcacaa gctggagtac aactcagtaa tctccaaggc aaagatacgc acagtgaaaa
     3181 tggttttggt tgttgtggct gccttcatta tctgctggac accaattcac atcttcatca
     3241 ttgtgaaaac actggttgac atcaaccaaa agaacccctt cgtgattgct agctggcact
     3301 tgtgcatcgc acttggttat accaacagca gcctcaatcc tgtgctttat gctttcttgg
     3361 atgagaattt caagagatgc ttccgagact tttgcctacc attcagaaca agggctgacc
     3421 agagtaatct gaaccgagca aggaatgcca caagggagcc agtttcagtt tgtgcccctt
     3481 cggatacagg aaagaagccg gtatgagaat tcctgcagcc cgggggatcc actagttcta
     3541 gagcggccgc ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     3601 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     3661 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     3721 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     3781 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     3841 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3901 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3961 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     4021 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct
     4081 cgacctggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     4141 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4201 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4261 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4321 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4381 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4441 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4501 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4561 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4621 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4681 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4741 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4801 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4861 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4921 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4981 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5041 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5101 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5161 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5221 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5281 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5341 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5401 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5461 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5521 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5581 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5641 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5701 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5761 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5821 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5881 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5941 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6001 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6061 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6121 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6181 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6241 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6301 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6361 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6421 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6481 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     6541 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     6601 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6661 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6721 cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg
     6781 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     6841 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     6901 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtacct agtaatcaat
     6961 tacggggtca ttagttcata gcccatatat ggagttccg
//
