LOCUS       sequence-238295-        6575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control to test technique on a target that is known to work.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6575)
  AUTHORS   Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, 
            Weiss TJ, Kambakam S, Vollbrecht MK, Lan M, McKeighan KC, Levey J, 
            Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien CB, 
            Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity BS, Solin SL, 
            Carlson DF, Dobbs DL, McGrail M, Essner J
  TITLE     Efficient targeted integration directed by short homology in 
            zebrafish and mammalian cells.
  JOURNAL   Elife. 2020 May 15;9. pii: 53968. doi: 10.7554/eLife.53968.
  PUBMED    32412410
REFERENCE   2  (bases 1 to 6575)
  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: "Elife."; 
            date: "2020-05-15"; volume: "9. pii: 53968. doi"; pages: " 
            10.7554/eLife.53968"
COMMENT     Sequence Label: Control-cx43.4-48H-pPRISM
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(271..282,283..445)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             572..628
                     /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             635..1345
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3219..3239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3243..3245,3246..3248,3249..3962)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3243..3245,3246..3248,3249..3959)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3288..3309)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3549..3568)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3896..3917
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3967..4191
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4261..4342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4466..4547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4800..4819
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4800..4818
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4841..5028,5029..5040)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5094..5113)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5105..5914
                     /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     complement(5172..5191)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6057..6575,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6546..6565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcggc ccgttttctt acgcggttgt tggatgaaat
      541 ctccaaccac tccaccttcg tgcttcccgg agccacgaac ttctctctgt taaagcaagc
      601 aggagacgtg gaagaaaacc ccggtcctct cgaggtgtct aagggcgagg aactgattaa
      661 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg
      721 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
      781 cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag
      841 cagaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccctga
      901 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
      961 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     1021 cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcca acaccgagat
     1081 gctgtacccc gctgacggcg gcctggaagg cagaagcgac atggccctga agctcgtggg
     1141 cgggggccac ctgatctgca acttcaagac cacatacaga tccaagaaac ccgctaagaa
     1201 cctcaagatg cccggcgtct actatgtgga ccacagactg gaaagaatca aggaggccga
     1261 caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag
     1321 caaactgggg cacaaactta attgaaacta gtaattaagt ctcagccacc gttaactgaa
     1381 catgtcaaaa cctgtggaga ctgttgagat ttgatgttct gaaaagataa agcctataaa
     1441 taaaatgttg cccaaatttc ctgcctgatg tttttctttg tctttgctac atggctttgc
     1501 tgctcggatc ggctcactct gtgtatgcca cgttcacttt gtactctcct tctcacggta
     1561 ggtttattat ttttagatgt gcagttagtt tctgtgaaat aacacaccac acactgatat
     1621 tgtctgtgca ttgacttggt gagtgcacat tgtttttgat cttgacatat ttatatttga
     1681 ttgatcaggt gaactgtgtg aatctaaagt gctccataca gatgttctgc attgaaaata
     1741 ttctcatttt attagtggaa gtgagtgtat gccacatcca atcaatttca gcaaacaccc
     1801 cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg cattactact ttgcagttaa
     1861 atatttaaca tttattccaa cacgaaaaaa agcagtaaat aacactttga caaacacgtc
     1921 aggacatctt atttttgtca ccctcacagg caatttagta taatatatta tatatatata
     1981 tatatcatat aataatattc agtataatat atatatatat atcatattat aatattcagt
     2041 ataatataaa acacaaacac atatatgtat aatataatat aacattttta tttattgaga
     2101 tgcctctatg gaccgtgtta taagaagtaa agatcaggag aagtaaacat gaagtgtaat
     2161 tatgaatact gatgttaaat taagctatga tgagttttca ctgttaattt accatctcaa
     2221 ttaaatgttg atgcctccat gaccaagtta agcagatgag actgaggaca actgtagaag
     2281 acaagatgtt cactttgctg aatatagctg gcttgacagt tatctatgac tctataaata
     2341 tatatatatt ttttttttat aaaatgattt atttataact atatatccat ttctcagaca
     2401 ggtgcttcat atccctcact cccgtagctg tccatgctgg atctgtcccc gttgttttta
     2461 aaaagctaaa taagttatta acatgactgc atccagcgag ccaaacctgt ctggtgtaca
     2521 gctaccagag aagcttgaga tcctagtcac cggcgttcct agttctttaa actgttacaa
     2581 agtgtttgga ccagcaactg aattgcactg taaattacca catcagggtc tccggggtca
     2641 acgctgctaa gcaatggact ggaaaatttg tgacattgag tttttggaat aactgggaat
     2701 gctggataac tctttgggta aagctatgga acaaaagtac cagcatgaac taagaaatga
     2761 ctgtcacagc acaacatcaa acacaatgca agacaaaggt tctctgcttt tgccttactt
     2821 gacagaaaaa gtaagctcta ggttcatcgc atgcagacag cagcaataca tgacatatat
     2881 aaaggctggc tcttttctcc cttccattga actgaaactt ccactcagtc agacttgcgg
     2941 gggatccaca gcctgactaa ctcgacaaac ccccccaaac ctaaggtgag ttgatcttta
     3001 agctttttac attttcagct cgcatatatc aattcgcgcg cattgtcaca ctagcaccca
     3061 ctagcggtca gactgcagat tgcagcacga aacaggaagc tgactccaca tggtcacatg
     3121 ctcactgaag tgttgacttc cctgacagct gtgcactttc taaaccggtt ttctcattca
     3181 tttacagttc agccggaatt cacctcgaga ccatggctcc aaagaagaag cgtaaggtaa
     3241 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     3301 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     3361 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3421 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     3481 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     3541 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     3601 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     3661 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     3721 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     3781 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3841 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     3901 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     3961 aaagatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4021 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4081 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4141 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     4201 cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca caactagaat
     4261 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4321 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4381 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattgatg
     4441 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4501 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4561 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4621 acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc gatgcatggg
     4681 agctctgacg tacgttgtct agcaaggaag tgggcaagat atggctcacg ttattcatca
     4741 tcttccgcat tgttttgaaa accgctgtgg cccgaacgcc tcccgctagc gatgggatct
     4801 aatacgactc actataggga gaccaaggga aaatagaatg aagtgatctc caaaaaataa
     4861 gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa
     4921 tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc
     4981 ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg
     5041 ggatccagct gcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     5101 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     5161 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     5221 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     5281 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     5341 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     5401 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     5461 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     5521 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5581 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5641 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5701 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5761 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5821 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5881 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     5941 ctggcaaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6001 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6061 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6121 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6181 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6241 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6301 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6361 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6421 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga
     6481 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6541 ccagggggaa acgcctggta tctttatagt cctgt
//
