LOCUS       sequence-227755-        4803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4803)
  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 4803)
  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: pGTag-NLS-TagRFP-B-actin
FEATURES             Location/Qualifiers
     source          1..4803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    636..657
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(672..677,678..695,696..702)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    710..726
                     /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)."
     primer_bind     715..737
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     734..750
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             746..1120
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSYGSKEEA"
     primer_bind     769..789
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        771..789
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(837..855)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(865..881)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1041..1097
                     /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             1104..1124
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1125..1838
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     promoter        complement(3045..3063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3068..3084)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3186..3205)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3197..4006
                     /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(3264..3283)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4153..4741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4642..4661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggatgt
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc gggaggcgtt
      601 cgggccacag cggacacgca ggtgcgcaac gcaattaatg tgagttagct cactcattag
      661 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      721 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      781 cactaaaggg aacaaaagct ggaggacctc gagggggggc ccggtaccca attcgcccta
      841 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
      901 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
      961 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1021 gacctgccac aggatccgga gccacgaact tctctctgtt aaagcaagca ggagacgtgg
     1081 aagaaaaccc cggtcctatg gctccaaaga agaagcgtaa ggtaatggtg tctaagggcg
     1141 aggaactgat taaggagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc
     1201 accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     1261 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct
     1321 tcatgtacgg cagcagaacc ttcatcaacc acacccaggg catccccgac ttctttaagc
     1381 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     1441 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     1501 gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     1561 ccaacaccga gatgctgtac cccgctgacg gcggcctgga aggcagaagc gacatggccc
     1621 tgaagctcgt gggcgggggc cacctgatct gcaacttcaa gaccacatac agatccaaga
     1681 aacccgctaa gaacctcaag atgcccggcg tctactatgt ggaccacaga ctggaaagaa
     1741 tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact
     1801 gcgacctccc tagcaaactg gggcacaaac ttaattgagg cgcgccacgg actgttacca
     1861 cttcacgccg actcaaactg cgcagagaaa cttcaaacga caacattggc atggcttttg
     1921 ttatttttgg cgcttgactc aggatctaaa aactggaacg gtgaaggtga cggcaatgtt
     1981 ttttggcaaa taagcatccc cgaagttcta caatgcatct gaggactcca tgttttgttt
     2041 tctttagtca ttccaaatgt ttgttaaatg cattgttccg aaacttattt gcctctatga
     2101 aggctgccca gtaattggga gcatacttaa cattgtagta ttgtatgtaa attatgtaac
     2161 aaaacaatgt ctgggttttt gtacttttca gccttaaaat cttgggttta tttttttttc
     2221 tttgttccaa aaaaacgaag ctttaccatt caagatgtaa aggtttccat tccccctggg
     2281 catattgtaa aagctgtgtg gaacgtggcg gtgccagaca tttggtgggg ccaacctgta
     2341 cactgactaa ttcaattcca ataaaagtgc acatgtaaga catcctaccc ctgtctcatt
     2401 tcctgtcttg gctctgagtg gcttcaataa aaatggcttt tgtgaagttg caccatgtta
     2461 attacatttc agcactttca caccaggttt ttgactaatt tgaatttatc gaagtttgac
     2521 cttttttttt ttttttttct tcaagctaat ttgaggttat caaaccattt cattggagga
     2581 aacttaatgt ttaaaagcat aaattcgaaa catgattcat ttatttaact gcttaatggt
     2641 gaaacaaact tcaagtaata caccaagtgt tccctcatat gaaggtagct aactgcaaac
     2701 ggccttaact ttccatctta agttgagcta cataaaggct ttaacaggaa ggaactgcca
     2761 gtttaaaagt caaaccacca tgactgatat aaattcttgt ccatataacc ttgcgtttca
     2821 cacaatatag ctatctactg ctaaataaat taccatggcg ttgtctagca aggaagtgaa
     2881 gagctatccg gatatatatg tatgttatca taactgtact ggctatgtca ggtggtaatg
     2941 actgtaacgt tacgttactc gttgtaggca cgacattgaa tatgctcttc tccgctgtgg
     3001 cccgaacgcc tcccgctagc gatgggatct cggccgcgat atcccctata gtgagtcgta
     3061 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     3121 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     3181 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     3241 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     3301 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     3361 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     3421 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     3481 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     3541 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     3601 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     3661 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     3721 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     3781 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     3841 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     3901 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     3961 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     4021 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     4081 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     4141 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4201 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4261 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4321 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4381 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4441 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4501 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4561 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4621 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4681 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4741 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4801 gtt
//
