LOCUS       sequence-189690-        3906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a HR repair template for knock-in of an EGFP insert that 
            fused to the 5'-end of human SFPQ gene, without disturbing its 
            promoter. Best used with px330-GFP-SFPQ vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3906)
  AUTHORS   Li R, Harvey AR, Hodgetts SI, Fox AH
  TITLE     Functional dissection of NEAT1 using genome editing reveals 
            substantial localisation of the NEAT1_1 isoform outside 
            paraspeckles.
  JOURNAL   RNA. 2017 Mar 21. pii: rna.059477.116. doi: 10.1261/rna.059477.116.
  PUBMED    28325845
REFERENCE   2  (bases 1 to 3906)
  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: "RNA. 2017 
            Mar 21. pii: rna.059477.116. doi: 10.1261/rna.059477.116."
COMMENT     Sequence Label: pUC57-GFP-SFPQ-RT
FEATURES             Location/Qualifiers
     source          1..3906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(join(736..1446,1447..1449,1450..1452))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(736..1446,1447..1449,1450..1452))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(778..799)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1127..1146
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1386..1407
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1816..1832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1816..1832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1829..1851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1840..1856
                     /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(1864..1870,1871..1888,1889..1894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1909..1930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2047..2064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2218..2806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2298..2317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2984..3793)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3707..3726
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3785..3804
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tattgggtct gcggctgtgg atgcggcggc ggctgatgcg gtggcggctg
      481 ctgcggcggt ggctgctgcg gtggtggctg ttgctgctgt tggtgtggag gcggtggcgg
      541 gatcggaggc ttagggccgc tctggcccgg gccaggaccc atggggccgc gattctgatt
      601 gaggcccatg ccgggcggcg gagaacggaa gtcgtggagg ccgccgcggc cgccgcctcc
      661 tccacgcctg tggaagccac caccgccacc gccacgactc cggaaccgat cccgagacat
      721 tgcagccgct gcagccttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
      781 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
      841 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
      901 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
      961 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     1021 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     1081 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     1141 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     1201 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
     1261 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     1321 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     1381 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     1441 cttgctcacc atgtctgtgg tcaaggggcg gtcgaggcaa aagcgaagaa gacgctcagg
     1501 aaacgtggag gccaccttgc ttctcacaaa atggcggatg acacaggcgg cgcgccgcct
     1561 ttgtcctcgt cttatatagg ccagctggca cggccccgcc tactccacga tcgacagctc
     1621 caatagccaa ttgcaattac tatgcttgat gacactaagt tgcgaacctc tgatcattcc
     1681 tattggcccc tgggtgagag gcgggaggca tggggggact gaagtttcca gttggccagt
     1741 gagcgaagaa gattgggtat cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag
     1801 cttggtgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1861 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     1921 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1981 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2041 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2101 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2161 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2221 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2281 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2341 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2401 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2461 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2521 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2581 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2641 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2701 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2761 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2821 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2881 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2941 aagcccaatc tgaataatgt tacaaccaat taaccaattc tgattagaaa aactcatcga
     3001 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     3061 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     3121 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     3181 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     3241 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     3301 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     3361 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     3421 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     3481 atgctgtttt tccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     3541 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     3601 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     3661 gcttcccata caagcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     3721 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgac gtttcccgtt
     3781 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     3841 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acgggccaga
     3901 gctgca
//
