LOCUS       sequence-345080-        5563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an FKBPV-HA2-P2A-eGFP cassette at the 
            mouse NANOG locus. Degradation Tag (dTAG) system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5563)
  AUTHORS   Liu NQ, Maresca M, van den Brand T, Braccioli L, Schijns MMGA, 
            Teunissen H, Bruneau BG, Nora EP, de Wit E
  TITLE     WAPL maintains a cohesin loading cycle to preserve 
            cell-type-specific distal gene regulation.
  JOURNAL   Nat Genet. 2021 Jan;53(1):100-109. doi: 10.1038/s41588-020-00744-4. 
            Epub 2020 Dec 14.
  PUBMED    33318687
REFERENCE   2  (bases 1 to 5563)
  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"; doi: 
            "10.1038/s41588-020-00744-4"; journalName: "Nat Genet"; date: 
            "2021-01"; volume: "53"; issue: "1"; pages: "100-109"
COMMENT     Sequence Label: pNQL006-NANOG-FKBPV-HA2-P2A-eGFP targeting con...
FEATURES             Location/Qualifiers
     source          1..5563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..42
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     34..51
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     35..51
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     58..77
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        58..76
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             598..918
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             925..951
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             925..951
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             955..981
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             991..1047
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1048..1050,1051..1764)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1048..1050,1051..1761)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1090..1111)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1351..1370)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1698..1719
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2810..2828)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2849..2865)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2849..2865)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2862..2884)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2873..2889
                     /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(2897..2903,2904..2921,2922..2927))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2942..2963
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3080..3097)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3251..3839)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3331..3350)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4010..4801,4802..4870))
                     /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     4633..4652
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4871..4975)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5001..5456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5138..5159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5350..5369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa
       61 tacgactcac tatagggcga attgggtact gtgcggccat gctaatcttt tcagtatttc
      121 tgcagtttac atctttagag tccccttttc actcattgca ttaatcacct ctttcagaag
      181 ggctcagcac cagtggagta tcccagcatc cattgcagct atccccaggg ctatctggtg
      241 aacgcatctg gaagcctttc catgtggggc agccagactt ggaccaaccc aacttggagc
      301 agccagacct ggaccaaccc aacttggaac aaccagacct ggaccaaccc aacttggagc
      361 agccaggcct ggaccgctca gtcctggaac ggccagcctt ggaatgctgc tccgctccat
      421 aacttcgggg aggactttct gcagccttac gtacagttgc agcaaaactt ctctgccagt
      481 gatttggagg tgaatttgga agccactagg gaaagccatg cgcattttag caccccacaa
      541 gccttggaat tattcctgaa ctactctgtg actccaccag gtgaaatagg atccggagga
      601 gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc
      661 tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aagttgattc ctcccgggac
      721 agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa
      781 ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc
      841 tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg
      901 gagcttctaa aactggaagg cggctacccc tacgacgtgc ccgactacgc cggctatccg
      961 tatgatgtcc cggactatgc aggctccgga gcaacaaact tctctctgct gaaacaagcc
     1021 ggagatgtcg aagagaatcc tggaccggtg agcaagggcg aggagctgtt caccggggtg
     1081 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1141 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1201 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1261 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1321 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1381 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1441 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1501 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1561 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1621 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1681 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1741 ggcatggacg agctgtacaa gtaagactta cgcaacatct gagcttaaag tcagggcaaa
     1801 gccaggttcc ttccttcttc caaatatttt catatttttt ttaaagattt atttattcat
     1861 tatatgtaag tacactgtag ctgtcttcag acactccaga agagggcgtc agatcttgtt
     1921 acgtatggtt gtgagccacc atgtggttgc tgggatttga actcctgacc ttcggaagag
     1981 cagtcgggtg ctcttatcca ctgagccatc tcaccagccc ctggtttatt tttttaatta
     2041 ttatttgctt tttgtttatc aagacagggt ttctctgcat agctctaatt gtctttgaac
     2101 tagctctgca gaccagcctg gccttgaact cagagatctg cccacttatc tttgcctcct
     2161 gaatgctggg accaaaggtg gcataccacc acacctggca tatatattgt ttatttctat
     2221 ttctattttt attggtgcca gagcaaacct aggacttaga acatgctggg caccaactca
     2281 acttctgagc tctatttaca acttggtgtg ttagtgtatt tgtcttagtt ctgaatttgt
     2341 ccttttttta gtgttaactc taggctttgg agacagtgag gtgcatatac tctctccttc
     2401 ccaagaataa gtgcttgaac acccttaccc acgcccaccc acccatgcta gtcttttttc
     2461 ttagaagcgt gggtcttggt atacactgtg tcattttgag gggtgaggtt taaaagtata
     2521 tacaaagtat aacgatatgg tggctactct cgaggatgag acagaaggac caggagtttg
     2581 agggtagctc agatatgcaa taagttcaag gccaacctgt actatgttta aatagtaaga
     2641 cagcatctcg ataaaataat aaaactaaag tctcaacaaa ataaaagctt tcacctatta
     2701 aggtgcttgc ttgtccttgg agtcccccaa gagtaactgc tatgttaata tctgtagaaa
     2761 gatgagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     2821 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2881 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2941 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3001 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3061 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3121 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3181 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3241 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3301 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3361 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3421 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3481 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3541 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3601 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3661 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3721 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3781 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3841 acgatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3901 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3961 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4021 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4081 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4141 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4201 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4261 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4321 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4381 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4441 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4501 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4561 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4621 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4681 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4741 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4801 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4861 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4921 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4981 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5041 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5101 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5161 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5221 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5281 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5341 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5401 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5461 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     5521 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agt
//
