LOCUS       sequence-248094-        8149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the transactivation domain of mouse Nanog and mCherry via 
            t2A linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8149)
  AUTHORS   Dhaliwal NK, Abatti LE, Mitchell JA
  TITLE     KLF4 protein stability regulated by interaction with pluripotency 
            transcription factors overrides transcriptional control.
  JOURNAL   Genes Dev. 2019 Jun 20. pii: gad.324319.119. doi: 
            10.1101/gad.324319.119.
  PUBMED    31221664
REFERENCE   2  (bases 1 to 8149)
  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: "Genes Dev. 
            2019 Jun 20. pii: gad.324319.119. doi: 10.1101/gad.324319.119."
COMMENT     Sequence Label: Nanog-t2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     654..678
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2758..2811
                     /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             2818..3522
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWDASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2960..2978)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3228..3247
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3559..3578)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3603..3651
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3853..4281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3940..3959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4150..4171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4290..4310)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4295..4624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4475..4610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4537..4556
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4691..5485
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4745..4764)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5355..5374
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5661..5782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5698..5717)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5752..5771
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5831..5847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5831..5847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5844..5866)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5855..5871
                     /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(5879..5885,5886..5903,5904..5909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5924..5945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6062..6079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6233..6821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6313..6332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6992..7783,7784..7852))
                     /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     7615..7634
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7853..7957)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8032..8051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      661 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      721 caccgggacc gatccagcct ccggactcta gactctgaag acctgcctct tcaaggcagc
      781 cctgattctt ctaccagtcc caaacaaaag ctctcaagtc ctgaggctga caagggccct
      841 gaggaggagg agaacaaggt ccttgccagg aagcagaaga tgcggactgt gttctctcag
      901 gcccagctgt gtgcactcaa ggacaggttt cagaagcaga agtacctcag cctccagcag
      961 atgcaagaac tctcctccat tctgaacctg agctataagc aggtggggtc cttagaagtt
     1021 gctgtaatgt aacacaatga gactgacttg ctaatgaatc gaactaacgt ctggacgtct
     1081 ctctaactat cctatcctcc tatcctatgc gcctgttaaa atcataatta tgtgattgat
     1141 tgattgattg attatgtata agtacactat agctgtcttc agatacacca gaagagggca
     1201 tcagatctca ttacggatgg ttgtgagcca ccatgtggtt gctgggattt gaactcagga
     1261 ccttcggaag agcagtcagt gctcttaacc gctgagccat ctctccagcc ccaattatgt
     1321 gatttatata tttcccagtt tgtttcattg gttactttat cttctacttc acctcccaca
     1381 cccatcccca aatcagggtc tcactgtagc cctgactggt ctctgtagac caggctggga
     1441 gaggttcacc tgcctctgtc tcctgggtgc tggaaataaa ggtgtgcctg gcttttttcc
     1501 tttcttttct tttttaagat tgtttatttt taaattcctt tagttcattg gtgttttgcc
     1561 tgcatgtgtt tgtctttgtg agagtatcag atctcctgga ctagagttac agacagtcgt
     1621 gagctgccat gtggccgctg agaattgaac ccaggtcctc tggaagagca gccagtgctc
     1681 ttaacagaag agccatttcc cccagcacct gtctttttca agaaagagtt tcactttaca
     1741 ggcctggctg tcatgtttaa ggtcgggctg tagacctgtc tgcattctgt atgtcttagc
     1801 ttgttcgaat gtcctgtcac tctgcaggtt aagacctggt ttcaaaacca aaggatgaag
     1861 tgcaagcggt ggcagaaaaa ccagtggttg aagactagca atggtctgat tcaggtaaca
     1921 agatgttttc tctgctttcc tttttccccc tccatcttac catcttattt tatgtgtctg
     1981 tgccgtgtgt gtgtgtgtgt gtgtgtgtgt ggctgaccta tggctgttgg gtatggatcc
     2041 cagatttcct gtaagagcag tcagtggctc tgcctttgct tcccagtgtc atcctgatcc
     2101 atctatgtcc attagtcctg tccctttccc atccctcttc ctccacgcac tctttcttgt
     2161 gatctttggt tctttgaggc aggctctctc tatgtagccc tggcagtcag agatctgcct
     2221 gcctagtctc aggagtgctg gggttaacgg cctgtgcggc catgctaatc ttttcagtat
     2281 ttctgcagtt tacatcttta gagtcccctt ttcactcatt gcattaatca cctctttcag
     2341 aagggctcag caccagtgga gtatcccagc atccattgca gctatcccca gggctatctg
     2401 gtgaacgcat ctggaagcct ttccatgtgg ggcagccaga cttggaccaa cccaacttgg
     2461 agcagccaga cctggaccaa cccaacttgg aacaaccaga cctggaccaa cccaacttgg
     2521 agcagccagg cctggaccgc tcagtcctgg aacggccagc cttggaatgc tgctccgctc
     2581 cataacttcg gggaggactt tctgcagcct tacgtacagt tgcagcaaaa cttctctgcc
     2641 agtgatttgg aggtgaattt ggaagccact agggaaagcc atgcgcattt tagcacccca
     2701 caagccttgg aattattcct gaactactct gtgactccac caggtgaaat agctagcgag
     2761 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc tggtaccgtg
     2821 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     2881 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     2941 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     3001 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     3061 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     3121 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     3181 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     3241 aagaagacca tgggctggga tgcctcctcc gagcggatgt accccgagga cggcgccctg
     3301 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     3361 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     3421 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     3481 gagggccgcc actccaccgg cggcatggac gagctgtaca agaccggtta gtaatgagtt
     3541 taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     3601 gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg
     3661 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     3721 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     3781 tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta
     3841 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3901 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3961 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4021 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4081 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4141 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4201 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4261 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4321 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4381 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4441 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4501 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4561 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4621 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     4681 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4741 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4801 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4861 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4921 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4981 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     5041 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     5101 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     5161 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     5221 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5281 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5341 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5401 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5461 tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag
     5521 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5581 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5641 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5701 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5761 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5821 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5881 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5941 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6001 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6061 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6121 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6181 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6241 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6301 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6361 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6421 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6481 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6541 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6601 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6661 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6721 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6781 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6841 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6901 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6961 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7021 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7081 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7141 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7201 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7261 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7321 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7381 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7441 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7501 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7561 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7621 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7681 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7741 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7801 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7861 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7921 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7981 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     8041 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     8101 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgac
//
