LOCUS       sequence-103220-        5990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for tdTomato knock-in via landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5990)
  AUTHORS   Ohtsuka M, Miura H, Mochida K, Hirose M, Hasegawa A, Ogura A, 
            Mizutani R, Kimura M, Isotani A, Ikawa M, Sato M, Gurumurthy CB
  TITLE     One-step generation of multiple transgenic mouse lines using an 
            improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).
  JOURNAL   BMC Genomics. 2015 Apr 9;16:274. doi: 10.1186/s12864-015-1432-5.
  PUBMED    25887549
REFERENCE   2  (bases 1 to 5990)
  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: "BMC 
            Genomics."; date: "2015-04-9"; volume: "16:274. doi"; pages: " 
            10.1186/s12864-015-1432-5"
COMMENT     Sequence Label: pBER
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    161..194
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(203..236)
                     /label=loxJTZ17
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      293..302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             299..1729
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    1856..1977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1893..1912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1947..1966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2355..3374)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(2355..3374)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     promoter        complement(3553..4052)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3601..3620)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3994..4017
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(4126..4173)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(4174..4207)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(4291..4307)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4291..4307)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      4355..4386
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        4387..4489
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4490..5305
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4563..4582)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5329..5356
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(5368..5955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5448..5467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcgg cctcggatcc gcgatcgatc cgcggtgcgg gtgccagggc gtgcccttgg
      181 gctccccggg cgcgtactcc acataacttc gtatagcata cattatagca atttatcgaa
      241 gttcctattc cgaagttcct attctatcag aagtatagga acttcaccgg tcgccaccat
      301 ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg
      361 ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
      421 cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
      481 cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc
      541 cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
      601 ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta
      661 caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac
      721 catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga
      781 gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat
      841 ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga
      901 catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg
      961 ccaccacctg ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc
     1021 ctcctccgag gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat
     1081 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1141 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     1201 ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga
     1261 catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1321 cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct
     1381 gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa
     1441 gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa
     1501 gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa
     1561 gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa
     1621 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga
     1681 gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagtaaa gcggccggcc
     1741 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     1801 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     1861 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1921 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     1981 cgtaaattgt aagcgttaat gatccactag aggatccccg ggtaccgagc tcgaattcac
     2041 tagcagttat acgccaagct ggcgcgccgc acacaaaaac caacacacag atcatgaaaa
     2101 taaagctctt ttattggtac cgaattccgc cagggagctc tcagactgtg gcagggaaac
     2161 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     2221 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     2281 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca tctagcggat
     2341 cccgctcacc tagcttcctt tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg
     2401 gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc gatttgtgta
     2461 cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg cgcccaagct
     2521 gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc aatgcggagc
     2581 atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc gaagtagcgc
     2641 gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc gacctcgtat
     2701 tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc
     2761 gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc
     2821 tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc
     2881 atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa atcacgccat
     2941 gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt ctggctaaga
     3001 tcggccgcag cgatcgcatc catggcctcc gcgaccggct gcagaacagc gggcagttcg
     3061 gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca ataggtcagg
     3121 ctctcgctga attccccaat gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag
     3181 tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac ccgcaggaca
     3241 tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc cgagagctgc
     3301 atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga cgtcgcggtg
     3361 agttcaggct tttttctagc agccatgcga aacgatcctc atcctgtctc ttgatcagat
     3421 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     3481 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccac
     3541 cgggctgcag gtcgaaaggc ccggagatga ggaagaggag aacagcgcgg cagacgtgcg
     3601 cttttgaagc gtgcagaatg ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct
     3661 gagcccgccc ctgagcccgc ccccggaccc accccttccc agcctctgag cccagaaagc
     3721 gaaggagcaa agctgctatt ggccgctgcc ccaaaggcct acccgcttcc attgctcagc
     3781 ggtgctgtcc atctgcacga gactagtgag acgtgctact tccatttgtc acgtcctgca
     3841 cgacgcgagc tgcggggcgg gggggaactt cctgactagg ggaggagtag aaggtggcgc
     3901 gaaggggcca ccaaagaacg gagccggttg gcgcctaccg gtggatgtgg aatgtgtgcg
     3961 aggccagagg ccacttgtgt agcgccaagt gcccagcggg gctgctaaag cgcatgctcc
     4021 agactgcctt gggaaaagcg cctcccctac ccggtagaat ctagtgaatt cgaagttcct
     4081 atactcctat aagaatagga acttcggaat aggaacttca tgatggaagt tcatatactt
     4141 tctagagaat aggaacttcg gaataggaac ttcataactt cgtataggat actttatacg
     4201 aagttatcag ctgccgctcg aggcgcgtga tcttacggca ttatacgtat gatcggtcca
     4261 cgatcagcta gattatctag tcagcttgat gtcatagctg tttcctgagg ctcaatactg
     4321 accatttaaa tcatacctga cctccatagc agaaagtcaa aagcctccga ccggaggctt
     4381 ttgacttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
     4441 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tgagccatat
     4501 tcaacgggaa acgtcttgct tgaagccgcg attaaattcc aacatggatg ctgatttata
     4561 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     4621 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     4681 tgttacagat gagatggtca ggctaaactg gctgacggaa tttatgcctc ttccgaccat
     4741 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tcccagggaa
     4801 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     4861 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacggcga
     4921 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttggtgcgag
     4981 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     5041 actcttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     5101 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     5161 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     5221 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     5281 cttgatgctc gatgagtttt tctaatgagg acctaaatgt aatcacctgg ctcaccttcg
     5341 ggtgggcctt tctgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5401 caaaaatcga tgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5461 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5521 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5581 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5641 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5701 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5761 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5821 tatctgcgct ctgctgaagc cagttacctc ggaaaaagag ttggtagctc ttgatccggc
     5881 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5941 aaaaaaggat ctcaagaaga tcctttgatt ttctaccgaa gaaaggccca
//
