LOCUS       sequence-358215-        5920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombineering cassette plasmid for T2A-tdTomato-FNF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5920)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5920)
  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: 
            "Unpublished"
COMMENT     Sequence Label: mT2A-tdTomato-FNF
FEATURES             Location/Qualifiers
     source          1..5920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             100..153
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             169..1599
                     /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"
     protein_bind    complement(1632..1665)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    1720..1944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1933..1950
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1982..2785)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2093..2112)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2703..2722
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2804..2851)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(2863..3362)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2911..2930)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3304..3327
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(3426..3459)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(3514..3532)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3553..3569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3553..3569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3566..3588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3577..3593
                     /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(3601..3607,3608..3625,3626..3631))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3646..3667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3784..3801)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3955..4543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4035..4054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4714..5505,5506..5574))
                     /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     5337..5356
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5575..5679)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5793..5812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
       61 ttggagattc gtttaaacgg aggatctggc ggaagatctg aaggccgcgg cagcctgctg
      121 acatgcggcg acgtggaaga gaaccctggc cctgctcctg gcagcacaat ggtgagcaag
      181 ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac
      241 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      301 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
      361 cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag
      421 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt
      481 ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag
      541 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg
      601 gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag
      661 gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc
      721 aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc
      781 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg
      841 ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag
      901 gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc
      961 atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1021 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
     1081 tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat
     1141 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1201 ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag
     1261 gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg
     1321 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
     1381 caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac
     1441 atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc
     1501 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac
     1561 cacctgttcc tgtacggcat ggacgagctg tacaagtaag gcgcgccgga tccattatgt
     1621 acctgactga tgaagttcct atactttcta gagaatagga acttcgaagg gttccgcaag
     1681 ctctagtcga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
     1741 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     1801 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     1861 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     1921 atggctggca actagaaggc acagtcgagg ctgatcagcg agctctagag aattgatccc
     1981 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     2041 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     2101 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     2161 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     2221 cgacgagatc atcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     2281 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     2341 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     2401 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     2461 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     2521 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     2581 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     2641 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     2701 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     2761 catcttgttc aatggccgat cccatggttt agttcctcac cttgtcgtat tatactatgc
     2821 cgatatacta tgccgatgat taattgtcaa caggctgcag gtcgaaaggc ccggagatga
     2881 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     2941 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     3001 accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt ggccgctgcc
     3061 ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga gactagtgag
     3121 acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg gggggaactt
     3181 cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg gagccggttg
     3241 gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt
     3301 gcccagcggg gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac
     3361 ccggtagaat ttcgacgacc tgcagccaag ctagcttggc tggacgtaaa ctcctcttca
     3421 gacctgaagt tcctatactt tctagagaat aggaacttcg gaattcgata tcaagcttat
     3481 cgatacctta attaagagct ccagcttttg ttccctttag tgagggttaa tttcgagctt
     3541 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3601 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3661 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3721 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3781 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3841 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3901 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3961 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4021 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4081 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4141 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4201 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4261 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4321 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4381 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4441 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4501 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4561 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4621 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4681 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4741 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4801 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4861 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4921 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4981 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5041 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5101 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5161 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5221 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5281 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5341 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5401 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5461 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5521 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5581 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5641 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5701 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5761 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5821 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5881 atttagtgct ttacggcacc tcgaccccaa aaaacttgat
//
