LOCUS       sequence-227500-        4125 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 4125)
  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 4125)
  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-SV40
FEATURES             Location/Qualifiers
     source          1..4125
                     /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"
     polyA_signal    1876..2010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1900..1919)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1954..1973
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2073..2168)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2367..2385)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2390..2406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2508..2527)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2519..3328
                     /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(2586..2605)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3475..4063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3964..3983
                     /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 cgcgcctcta gaactatagt
     1861 gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     1921 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1981 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2041 caggttcagg gggaggtgtg ggaggttttt tccaacttta ttatacaaag ttggcattat
     2101 aaaaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     2161 cattatttgg agctccatgg cgttgtctag caaggaagtg aagagctatc cggatatata
     2221 tgtatgttat cataactgta ctggctatgt caggtggtaa tgactgtaac gttacgttac
     2281 tcgttgtagg cacgacattg aatatgctct tctccgctgt ggcccgaacg cctcccgcta
     2341 gcgatgggat ctcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt
     2401 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     2461 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     2521 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     2581 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     2641 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     2701 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     2761 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     2821 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     2881 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     2941 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     3001 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     3061 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     3121 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     3181 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     3241 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     3301 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     3361 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     3421 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3481 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3541 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3601 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3661 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3721 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3781 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3841 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3901 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3961 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4021 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4081 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtt
//
