LOCUS       sequence-358194-        7168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombineering cassette plasmid for 
            T2A-TagRFP-T-linker-dre-PBD*-FNF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7168)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7168)
  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            "Unpublished"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-TagRFP-T-linker-dre-PBD*-FNF
FEATURES             Location/Qualifiers
     source          1..7168
                     /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..900
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     protein_bind    complement(2880..2913)
                     /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    2968..3192
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3181..3198
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3230..4033)
                     /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(3341..3360)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3951..3970
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4052..4099)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4111..4610)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4159..4178)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4552..4575
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(4674..4707)
                     /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(4762..4780)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4801..4817)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4801..4817)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4814..4836)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4825..4841
                     /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(4849..4855,4856..4873,4874..4879))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4894..4915
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5032..5049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5203..5791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5283..5302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5962..6753,6754..6822))
                     /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     6585..6604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6823..6927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7041..7060)
                     /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 ggtgtctaag
      181 ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtgaac
      241 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
      301 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
      361 agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc cgatttcttt
      421 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
      481 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
      541 atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
      601 gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag aaccgacatg
      661 gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacagatcc
      721 aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca cagactggaa
      781 agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga
      841 tactgcgacc tccctagcaa actggggcac aaacttaatg gcatggacga gctgtacaag
      901 ggaggatctg gcggccgctc tgccacaaac ttcagcctgc tgaagcaggc cggcgacgcc
      961 gctgaaaatc ctggccctgc tcctggaagc accatgggtg ctagcgagct gatcatctct
     1021 ggctcctctg gaggattcct gaggaacatc ggcaaggagt accaggaggc tgctgagaac
     1081 ttcatgagat tcatgaatga ccagggagcc tacgccccta acaccctgag agacctgagg
     1141 ctggtgttcc actcctgggc tagatggtgc cacgctagac agctggcctg gttccctatc
     1201 tctcctgaga tggctaggga gtacttcctt cagctgcacg atgctgacct ggcctctacc
     1261 accatcgaca agcactacgc catgctgaac atgctgctgt cccactgtgg cctgcctcct
     1321 ctgtctgatg acaagtctgt gagcctggcc atgaggagaa tccggagaga ggctgccacc
     1381 gagaagggag agagaaccgg ccaggccatc cctctgagat gggatgacct gaagctgctg
     1441 gatgtgctgc tgtctagatc tgagagactg gtggacctga ggaatagggc cttcctgttt
     1501 gtggcctaca acaccctgat gaggatgtct gagatctcta ggatcagagt gggagacctg
     1561 gaccagaccg gagacaccgt gaccctgcac atctcccaca ccaagaccat caccaccgct
     1621 gctggcctgg acaaagtgct gtctaggagg accaccgctg tgctgaatga ctggctggat
     1681 gtgtctggcc tgagagagca ccctgacgct gtgctgttcc ctcctatcca ccggagcaac
     1741 aaggctagga tcaccaccac ccctctgacc gcccctgcca tggagaagat ttttagcgat
     1801 gcctgggtgc tgctgaacaa gagggatgcc acccctaaca agggccgcta ccggacctgg
     1861 accggccact ctgctagagt gggagctgcc atcgacatgg ctgagaagca agtgtccatg
     1921 gtggagatca tgcaggaggg cacctggaaa aagcctgaga cactgatgag atacctgagg
     1981 aggggaggag tgtctgtggg agccaactct aggctgatgg actccgctag cggcgccctg
     2041 gatgctgttg ctctcccaca gccagtgggc gttccaaatg aaagccaagc cctaagccag
     2101 agattcactt tttcaccagg tcaagacata cagttgattc caccactgat caacctgtta
     2161 atgagcattg aaccagatgt gatctatgca ggacatgaca acacaaaacc tgacacctcc
     2221 agttctttgc tgacaagtct taatcaacta ggcgagaggc aacttctttc agtagtcaag
     2281 tggtctaaat cattgccagg ttttcgaaac ttacatattg atgaccagat aactctcatt
     2341 cagtattctt ggatgagctt aatggtgttt ggtctaggat ggagatccta caaacacgtc
     2401 agtgggcaga tgctgtattt tgcacctgat ctaatactaa atgaacagcg gatgaaggaa
     2461 tcatcattct attcattatg ccttaccatg tggcagatcc cacaggagtt tgtcaagctt
     2521 caagttagcc aagaagagtt cctctgtatg aaagtattgt tacttcttaa tacaattcct
     2581 ttggaagggc tacgaagtca aacccagttt gaggagatga ggtcaagcta cattagagag
     2641 ctcatcaagg caattggttt gaggcaaaaa ggagttgtgt cgagctcaca gcgtttctat
     2701 caacttacaa aacttcttga taacttgcat gatcttgtca aacaacttca tctgtactgc
     2761 ttgaatacat ttatccagtc ccgggcactg agtgttgaat ttccagaaat gatgtctgaa
     2821 gttattgcta tgcatgcgta cggatagggc gcgccggatc cattatgtac ctgactgatg
     2881 aagttcctat actttctaga gaataggaac ttcgaagggt tccgcaagct ctagtcgagc
     2941 cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc
     3001 tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat
     3061 gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag
     3121 tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac
     3181 tagaaggcac agtcgaggct gatcagcgag ctctagagaa ttgatcccct cagaagaact
     3241 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     3301 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     3361 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     3421 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcat
     3481 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     3541 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     3601 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     3661 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     3721 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     3781 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     3841 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     3901 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     3961 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     4021 tggccgatcc catggtttag ttcctcacct tgtcgtatta tactatgccg atatactatg
     4081 ccgatgatta attgtcaaca ggctgcaggt cgaaaggccc ggagatgagg aagaggagaa
     4141 cagcgcggca gacgtgcgct tttgaagcgt gcagaatgcc gggcctccgg aggaccttcg
     4201 ggcgcccgcc ccgcccctga gcccgcccct gagcccgccc ccggacccac cccttcccag
     4261 cctctgagcc cagaaagcga aggagcaaag ctgctattgg ccgctgcccc aaaggcctac
     4321 ccgcttccat tgctcagcgg tgctgtccat ctgcacgaga ctagtgagac gtgctacttc
     4381 catttgtcac gtcctgcacg acgcgagctg cggggcgggg gggaacttcc tgactagggg
     4441 aggagtagaa ggtggcgcga aggggccacc aaagaacgga gccggttggc gcctaccggt
     4501 ggatgtggaa tgtgtgcgag gccagaggcc acttgtgtag cgccaagtgc ccagcggggc
     4561 tgctaaagcg catgctccag actgccttgg gaaaagcgcc tcccctaccc ggtagaattt
     4621 cgacgacctg cagccaagct agcttggctg gacgtaaact cctcttcaga cctgaagttc
     4681 ctatactttc tagagaatag gaacttcgga attcgatatc aagcttatcg ataccttaat
     4741 taagagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
     4801 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4861 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4921 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4981 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5041 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5101 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5161 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5221 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5281 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5341 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5401 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5461 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5521 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5581 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5641 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5701 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5761 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5821 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5881 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5941 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6001 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6061 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6121 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6181 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6241 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6301 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6361 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6421 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6481 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6541 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6601 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6661 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6721 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6781 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6841 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6901 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     6961 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     7021 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     7081 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     7141 acggcacctc gaccccaaaa aacttgat
//
