LOCUS       sequence-416525-        5163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template for mouse Mettl3 
            knockout and reporter knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5163)
  AUTHORS   Leoni C, Bataclan M, Ito-Kureha T, Heissmeyer V, Monticelli S
  TITLE     The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability 
            and limits functional responses in mast cells.
  JOURNAL   Nat Commun. 2023 Jun 29;14(1):3862. doi: 10.1038/s41467-023-39614-y.
  PUBMED    37386028
REFERENCE   2  (bases 1 to 5163)
  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 
            Commun."; date: "2023-06-29"; volume: "14(1):3862. doi"; pages: " 
            10.1038/s41467-023-39614-y"
COMMENT     Sequence Label: pBluescript II Ks(+)-ZsGreen1-SV40 PolyA-Stop ...
FEATURES             Location/Qualifiers
     source          1..5163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1336..1352
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1336..1352
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1366..2061
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     polyA_signal    2068..2202
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2105..2124)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2159..2178
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2975..2995)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2975..2993)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3014..3030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3014..3030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3027..3049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3038..3054
                     /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(3062..3068,3069..3086,3087..3092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3107..3128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3245..3262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3416..4004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3496..3515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4175..4966,4967..5035))
                     /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     4798..4817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5036..5140)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgcagga ttgtgagttg cagaaccaca tagtgaaact ccactctcct
      721 ttcacacact ttgtatagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa
      781 agaaagaaag aaagaaagaa agaaagaagg agggagggag ggagggagga aggaaggaag
      841 gaagataata tttaacttag aaaatctggg gccttaaggg cagaaatcta tgatacagtg
      901 cttgaccgta tgtgcaagga ccagaatata caatcaacca gactaacagt ctgttcactc
      961 cttcccgtcc atccatctct ccacgcatac acccacccgt caaacactca cacttgttct
     1021 agccctgcta ttttaaatgc gttgtcacta acgacttaaa gaaacaacat ggcgggtctg
     1081 ccagctacca acatggcaaa cacagcctca gcgctcccgg aaacacgaga gagaactacc
     1141 gtgaggatta cgtccggctg gttcgccaac cggaagtggc ttttcatctt ggctctatcc
     1201 ggctggcctt cgggcgtcct cgtgagaatt agatgtttct tagttctagg ctggggaggt
     1261 gctagtccgc cgcgcctgat tcgagccgag tccgcgctgg gagctaggat gtcggacacg
     1321 tggagctcta tcccatctag aactagtgga tcccccgcaa tacccatggc ccagtccaag
     1381 cacggcctga ccaaggagat gaccatgaag taccgcatgg agggctgcgt ggacggccac
     1441 aagttcgtga tcaccggcga gggcatcggc taccccttca agggcaagca ggccatcaac
     1501 ctgtgcgtgg tggagggcgg ccccttgccc ttcgccgagg acatcttgtc cgccgccttc
     1561 atgtacggca accgcgtgtt caccgagtac ccccaggaca tcgtcgacta cttcaagaac
     1621 tcctgccccg ccggctacac ctgggaccgc tccttcctgt tcgaggacgg cgccgtgtgc
     1681 atctgcaacg ccgacatcac cgtgagcgtg gaggagaact gcatgtacca cgagtccaag
     1741 ttctacggcg tgaacttccc cgccgacggc cccgtgatga agaagatgac cgacaactgg
     1801 gagccctcct gcgagaagat catccccgtg cccaagcagg gcatcttgaa gggcgacgtg
     1861 agcatgtacc tgctgctgaa ggacggtggc cgcttgcgct gccagttcga caccgtgtac
     1921 aaggccaagt ccgtgccccg caagatgccc gactggcact tcatccagca caagctgacc
     1981 cgcgaggacc gcagcgacgc caagaaccag aagtggcacc tgaccgagca cgccatcgcc
     2041 tccggctccg ccttgccctg actgcagaac ttgtttattg cagcttataa tggttacaaa
     2101 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2161 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctccatcaa ggaattcacg
     2221 attgttggtt accccatctt tcccgagtag ctgaaacgca tgcccacctg cgggaaagct
     2281 tgcagtgcct cctcccgggc gtcggttttt caagcgtaac accacaccaa catgggtcca
     2341 ctcctctcct tgggtcccat tccagtcctg tctaggagaa tgttgcgtcc gtgaagcctc
     2401 tcagtacctt attccgagag ccagcaaaac ccgttttgtt gcttctgtgc ctgttttacc
     2461 ttagttgtaa cgtttgcttt gtcactgtct tttctttccc atgtgggcgg ggagcaagga
     2521 ttgcttctgt atatctatcc ccagagcccg gtttagcagc cacaacaggg tcctcactag
     2581 gaccttctca ctaggacctt aatgaatgag tagtgcaagt gtttacaaac gtagcaagaa
     2641 tctgaaaaca cttgattttg gtctttaaac attttccccc tcccccttgt atgacaaagt
     2701 tagaaaaatt gaaggcactt aaaagagtac taccctaata gccttctcca gcacaagctc
     2761 aattatttaa ggtaaaccag ttgcaaagct ctgcgagaat accgaatcca tactacatgt
     2821 gtgggtagca tgtcagtgct ttgcagtatt cacagctcac aacagcagac cttggtgaaa
     2881 ttcagtctca ccgtaatttg tgaacattta ttgcatatct acactactca tgggtttcgg
     2941 cctcgagggg gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct
     3001 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3061 acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3121 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3841 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5041 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5101 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5161 cac
//
