LOCUS       sequence-243948-        7390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of a non-conducting mutant of Kir2.1 
            (FLAG-tagged) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7390)
  AUTHORS   Fujimoto S, Leiwe MN, Aihara S, Sakaguchi R, Muroyama Y, Kobayakawa 
            R, Kobayakawa K, Saito T, Imai T
  TITLE     Activity-dependent local protection and lateral inhibition control 
            synaptic competition in developing mitral cells in mice.
  JOURNAL   Dev Cell. 2023 Jul 24;58(14):1221-1236.e7. doi: 
            10.1016/j.devcel.2023.05.004. Epub 2023 Jun 7.
  PUBMED    37290446
REFERENCE   2  (bases 1 to 7390)
  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: 
            "10.1016/j.devcel.2023.05.004"; journalName: "Dev Cell"; date: 
            "2023-07-24- 24"; volume: "58"; issue: "14"; pages: "1221-1236.e7"
COMMENT     Sequence Label: pCALNL-mutKir2.1
FEATURES             Location/Qualifiers
     source          1..7390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1753..1786)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1836..2630
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1890..1909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2500..2519
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2810..2944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2958..2991)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3025..3034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4321..4344
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4433..4452)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4498..4553
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4552..4571)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4914..4930)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4914..4930)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4927..4949)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4938..4954
                     /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(4962..4968,4969..4986,4987..4992))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5007..5028
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5113..5247
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5267..5401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5469..5486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5640..6228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5720..5739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6399..7190,7191..7259))
                     /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     7022..7041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7260..7364)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgattaattc
     1741 gagcgaacgc gtataacttc gtatagcata cattatacga agttatctcg agtcggattt
     1801 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     1861 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     1921 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     1981 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     2041 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     2101 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     2161 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     2221 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     2281 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     2341 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     2401 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     2461 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     2521 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     2581 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     2641 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     2701 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     2761 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccatcgata acttgtttat
     2821 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2881 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2941 gatcaatccg aacgcgtata acttcgtata gcatacatta tacgaagtta tctcgagtcg
     3001 ctcggtacga tttaaattga attcgccacc atgggcagtg tgagaaccaa ccgctacagc
     3061 atcgtctctt cggaggaaga tggcatgaag ctggccacta tggcagttgc caatggcttt
     3121 gggaatggca agagtaaagt ccatacccga caacagtgca ggagccgctt tgtgaagaaa
     3181 gatggtcatt gcaatgttca gtttatcaac gtgggtgaga agggacagag gtacctggca
     3241 gacatcttta ctacctgtgt cgacatccgc tggaggtgga tgctggttat cttctgtctt
     3301 gccttcgtgc tctcctggct gttctttggc tgtgtgtttt ggttgatagc cctgctccat
     3361 ggggatctag atacttctaa agtgagcaaa gcatgcgtgt cagaggtcaa cagcttcacg
     3421 gctgccttcc tcttctccat cgagacccag acaaccattg ccgctgcttt caggtgtgtg
     3481 acagacgagt gcccaattgc tgtcttcatg gtggtattcc agtcaatcgt aggctgcatc
     3541 attgacgcct tcatcattgg tgcagtcatg gcgaagatgg caaagccaaa gaagagaaat
     3601 gagactcttg tcttcagtca caatgctgtg attgccatga gggatggcaa actctgcttg
     3661 atgtggagag tgggtaacct tcgaaagagc caccttgtgg aagctcatgt ccgggcacag
     3721 cttctcaaat ctaggatcac ttcagaaggg gagtatatcc ctttggacca gatagacatc
     3781 aatgttggtt ttgatagtgg aattgaccgc atatttctag tgtcccccat cactatcgtt
     3841 cacgaaatag atgaagacag ccctttatat gacttgagta agcaggacat tgacaatgca
     3901 gactttgaaa ttgttgtcat actggaaggc atggtggagg cgactgccat gacaactcaa
     3961 tgccggagtt cgtatctggc caatgaaatt ctctggggtc accgctatga gccagtgctc
     4021 tttgaagaga aacactacta taaagtagac tattcaagat tccataagac ttatgaagta
     4081 cctaacaccc ccctttgtag tgccagagac ttagcagaga agaaatacat cctttcaaat
     4141 gcaaattcat tttgctatga aaatgaagtt gccctaacaa gcaaagagga agaggaggat
     4201 agtgagaacg gagtcccaga gagcacaagc acagactcac ctcctggcat agatctccac
     4261 aaccaggcaa gcgtacctct agagcccagg cccttaaggc gagaatcgga gatactcgag
     4321 gactacaagg acgacgacga caagtgagcg gccgcactcc tcaggtgcag gctgcctatc
     4381 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     4441 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     4501 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     4561 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     4621 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     4681 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     4741 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     4801 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     4861 ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag
     4921 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4981 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5041 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt
     5101 cagcaaccat agtcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5161 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     5221 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc
     5281 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5341 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     5401 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5461 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5521 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5581 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5641 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5701 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5761 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5821 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5881 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5941 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6001 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6061 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6121 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6181 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6241 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6301 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6361 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6421 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6481 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6541 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6601 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6661 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6721 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6781 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6841 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6901 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6961 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7021 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7081 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7141 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7201 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7261 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7321 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7381 gtgccacctg
//
