LOCUS       sequence-358195-        5950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombineering cassette plasmid for T2A-td-sfGFP-FNF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5950)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5950)
  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            "Unpublished"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-td-sfGFP-FNF
FEATURES             Location/Qualifiers
     source          1..5950
                     /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             join(169..171,172..882)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             169..882
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(913..915,916..1629)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             913..1626
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    complement(1662..1695)
                     /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    1750..1974
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1963..1980
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2012..2815)
                     /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(2123..2142)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2733..2752
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2834..2881)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(2893..3392)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2941..2960)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3334..3357
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(3456..3489)
                     /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(3544..3562)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3583..3599)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3583..3599)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3596..3618)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3607..3623
                     /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(3631..3637,3638..3655,3656..3661))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3676..3697
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3814..3831)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3985..4573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4065..4084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4744..5535,5536..5604))
                     /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     5367..5386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5605..5709)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5823..5842)
                     /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 gagcaaagga
      181 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
      241 cacaaatttt ctgtccgtgg agagggtgaa ggtgatgcta caaacggaaa actcaccctt
      301 aaatttattt gcactactgg aaaactacct gttccgtggc caacacttgt cactactctg
      361 acctatggtg ttcaatgctt ttcccgttat ccggatcaca tgaaacggca tgactttttc
      421 aagagtgcca tgcccgaagg ttatgtacag gaacgcacta tatctttcaa agatgacggg
      481 acctacaaga cgcgtgctga agtcaagttt gaaggtgata cccttgttaa tcgtatcgag
      541 ttaaagggta ttgattttaa agaagatgga aacattcttg gacacaaact cgagtacaac
      601 tttaactcac acaatgtata catcacggca gacaaacaaa agaatggaat caaagctaac
      661 ttcaaaattc gccacaacgt tgaagatggt tccgttcaac tagcagacca ttatcaacaa
      721 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcgacacaa
      781 tctgtccttt cgaaagatcc caacgaaaag cgtgaccaca tggtccttct tgagtttgta
      841 actgctgctg ggattacaca tggcatggat gagctctaca aaggttctgc tggatctgca
      901 gcaggatctg gtatgagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
      961 gaattagatg gtgatgttaa tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat
     1021 gctacaaacg gaaaactcac ccttaaattt atttgcacta ctggaaaact acctgttccg
     1081 tggccaacac ttgtcactac tctgacctat ggtgttcaat gcttttcccg ttatccggat
     1141 cacatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc
     1201 actatatctt tcaaagatga cgggacctac aagacgcgtg ctgaagtcaa gtttgaaggt
     1261 gatacccttg ttaatcgtat cgagttaaag ggtattgatt ttaaagaaga tggaaacatt
     1321 cttggacaca aactcgagta caactttaac tcacacaatg tatacatcac ggcagacaaa
     1381 caaaagaatg gaatcaaagc taacttcaaa attcgccaca acgttgaaga tggttccgtt
     1441 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     1501 gacaaccatt acctgtcgac acaatctgtc ctttcgaaag atcccaacga aaagcgtgac
     1561 cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc
     1621 tacaaatgag gcgcgccgga tccattatgt acctgactga tgaagttcct atactttcta
     1681 gagaatagga acttcgaagg gttccgcaag ctctagtcga gccccagctg gttctttccg
     1741 cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct
     1801 cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat
     1861 gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca
     1921 aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg
     1981 ctgatcagcg agctctagag aattgatccc ctcagaagaa ctcgtcaaga aggcgataga
     2041 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     2101 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     2161 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     2221 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc atcgccgtcg ggcatgcgcg
     2281 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     2341 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     2401 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     2461 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     2521 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     2581 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     2641 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     2701 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     2761 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatggccgat cccatggttt
     2821 agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa
     2881 caggctgcag gtcgaaaggc ccggagatga ggaagaggag aacagcgcgg cagacgtgcg
     2941 cttttgaagc gtgcagaatg ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct
     3001 gagcccgccc ctgagcccgc ccccggaccc accccttccc agcctctgag cccagaaagc
     3061 gaaggagcaa agctgctatt ggccgctgcc ccaaaggcct acccgcttcc attgctcagc
     3121 ggtgctgtcc atctgcacga gactagtgag acgtgctact tccatttgtc acgtcctgca
     3181 cgacgcgagc tgcggggcgg gggggaactt cctgactagg ggaggagtag aaggtggcgc
     3241 gaaggggcca ccaaagaacg gagccggttg gcgcctaccg gtggatgtgg aatgtgtgcg
     3301 aggccagagg ccacttgtgt agcgccaagt gcccagcggg gctgctaaag cgcatgctcc
     3361 agactgcctt gggaaaagcg cctcccctac ccggtagaat ttcgacgacc tgcagccaag
     3421 ctagcttggc tggacgtaaa ctcctcttca gacctgaagt tcctatactt tctagagaat
     3481 aggaacttcg gaattcgata tcaagcttat cgatacctta attaagagct ccagcttttg
     3541 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     3601 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3661 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3721 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3781 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3841 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3901 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3961 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4021 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4081 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4141 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4201 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4261 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4321 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4381 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4441 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4501 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4561 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4621 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4681 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4741 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4801 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4861 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4921 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4981 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5041 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5101 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5161 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5221 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5281 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5341 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5401 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5461 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5521 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5581 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5701 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
     5761 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5821 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5881 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5941 aaaacttgat
//
