LOCUS       sequence-416545-        5412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template for mouse cKit 
            knockout and reporter knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5412)
  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 5412)
  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..5412
                     /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     1387..1403
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1387..1403
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1417..2112
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     polyA_signal    2119..2253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2156..2175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2210..2229
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3224..3244)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3224..3242)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3263..3279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3263..3279)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3276..3298)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3287..3303
                     /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(3311..3317,3318..3335,3336..3341))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3356..3377
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3494..3511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3665..4253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3745..3764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4424..5215,5216..5284))
                     /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     5047..5066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5285..5389)
                     /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 ggccgcgtat tatagtatat atatttatat aacatatgta tgtgtgcacg
      721 catgtgtgtg tgtatcatac ctgcctactg ctatcctgcc tcctcagtgc attcctgtcc
      781 tcctcttctc tagctctcct ccttcaccga gtctttctgt tttgcctttg gtgaccaatt
      841 gactctaata agtagggttt ggggcttggt tatttgagca agggtggctt accagtggct
      901 acaccactga ggaatacatc gtgcagctca tttttgtttc tggtttccac tgtgtaagac
      961 gtagctgtga tttttttttt aaaagatgaa tattcatgta acaacagcct gaggacagac
     1021 catcctcgtt gggcacggtt gacccagaga aagctgtgtc cagcccggca acttgtacac
     1081 caagcgtgac ccataggact aattgaaggc tgcagaaaat aactttatag atgaatttaa
     1141 actttagaca gaattccctt tgcagccaca aatagcaggg ctatttttat tgtagcatcc
     1201 acagtcgaga tggaactcaa tattagaaga aatggtttac ttcgccaaga cagctcacac
     1261 tcaacacagt ccttgttttt cttggcagcc acgtctcagc catctgcaag tccaggggag
     1321 ccgtctccgc catccatcca tccagcacaa tcagagttaa tagttgaagc tggcgacacc
     1381 ctcagttcta gaactagtgg atcccccgca atacccatgg cccagtccaa gcacggcctg
     1441 accaaggaga tgaccatgaa gtaccgcatg gagggctgcg tggacggcca caagttcgtg
     1501 atcaccggcg agggcatcgg ctaccccttc aagggcaagc aggccatcaa cctgtgcgtg
     1561 gtggagggcg gccccttgcc cttcgccgag gacatcttgt ccgccgcctt catgtacggc
     1621 aaccgcgtgt tcaccgagta cccccaggac atcgtcgact acttcaagaa ctcctgcccc
     1681 gccggctaca cctgggaccg ctccttcctg ttcgaggacg gcgccgtgtg catctgcaac
     1741 gccgacatca ccgtgagcgt ggaggagaac tgcatgtacc acgagtccaa gttctacggc
     1801 gtgaacttcc ccgccgacgg ccccgtgatg aagaagatga ccgacaactg ggagccctcc
     1861 tgcgagaaga tcatccccgt gcccaagcag ggcatcttga agggcgacgt gagcatgtac
     1921 ctgctgctga aggacggtgg ccgcttgcgc tgccagttcg acaccgtgta caaggccaag
     1981 tccgtgcccc gcaagatgcc cgactggcac ttcatccagc acaagctgac ccgcgaggac
     2041 cgcagcgacg ccaagaacca gaagtggcac ctgaccgagc acgccatcgc ctccggctcc
     2101 gccttgccct gactgcagaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2161 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2221 aaactcatca atgtatctta tcatgtctgg atctccatca aggaattcaa cacatacacg
     2281 tgcagcaaca gcaatggcct cacgagttct atttacgtgt ttgttagagg taaatgctgg
     2341 gttttccgtg gcgctgtggt ggagggtagt tgcttccctg ttctcaccca gaggtgacca
     2401 aaccacaggc aggatttggc tggctggttc aaggaggcct aacatacatg gttttcttca
     2461 ccagcttcct catggcttgg agtgtgaccc ctgctggggg tctgcaggcc ggaactggat
     2521 tacagtctgg ttaacccatt tgtcctggtg ctcggagtca accccgggaa taaattaggc
     2581 cctttgggaa aactgtattt taatgccttg aaatactaaa tattcccact taagtaaaaa
     2641 cacttcaggg agcccaaggc ctcaaaaggc ccggacagac tagtacttca aaagacttgg
     2701 cccactctta gggactacat tccagaaaac aactcggaca gctaagcatt tgttgtcagt
     2761 cataagctcc ttcggggttg gttcttttca gcaattacag cctctcagaa acagaagaca
     2821 aagatgtggc agttggagcc cctacacctc atttcctaga cgcattcagc aatctgtgga
     2881 tgtgtgtttg ctatcatgtc aagaactctc ggggcggtat cctttgggtg atgtaaaaac
     2941 aaagctaatg tcagtctaag agacaaatga gaaaggcaga agatggcagt cctggttgta
     3001 gcaccagttg gactgggact gggtctgtca ttgatcgcat gcttgcagct ctgtgagtct
     3061 aggctggatg ctttcctgtg cctgggaggt gctgcccggg gcctccgttt ctccctgtgg
     3121 tcctcagata agatgagcag tctcacttag ggtattgtta gggaaacaca attatacaaa
     3181 tcttagcacg ctcgaggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa
     3241 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3301 caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag
     3361 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3421 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3481 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3541 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3601 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3661 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3721 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3781 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3841 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3901 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3961 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4021 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4081 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4141 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4201 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4261 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4321 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4381 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4441 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4501 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4561 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4621 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4681 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4741 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4801 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4861 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4921 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4981 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5041 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5101 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5161 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5221 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5281 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5341 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5401 gaaaagtgcc ac
//
