LOCUS       sequence-227721-        4137 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 4137)
  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 4137)
  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-eGFP-SV40
FEATURES             Location/Qualifiers
     source          1..4137
                     /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             join(1125..1127,1128..1130,1131..1844)
                     /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(1125..1127,1128..1130,1131..1841)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1170..1191)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1431..1450)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1778..1799
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1888..2022
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1912..1931)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1966..1985
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2085..2180)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2379..2397)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2402..2418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2520..2539)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2531..3340
                     /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(2598..2617)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3487..4075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3976..3995
                     /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 agcaagggcg
     1141 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1201 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1261 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1321 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1381 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1441 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1501 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1561 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1621 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1681 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1741 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1801 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaactcgag ggcgcgcctc
     1861 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt
     1921 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     1981 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     2041 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttccaactt tattatacaa
     2101 agttggcatt ataaaaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag
     2161 tcaaaataaa atcattattt ggagctccat ggcgttgtct agcaaggaag tgaagagcta
     2221 tccggatata tatgtatgtt atcataactg tactggctat gtcaggtggt aatgactgta
     2281 acgttacgtt actcgttgta ggcacgacat tgaatatgct cttctccgct gtggcccgaa
     2341 cgcctcccgc tagcgatggg atctcggccg cgatatcccc tatagtgagt cgtattacat
     2401 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     2461 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     2521 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     2581 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     2641 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     2701 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     2761 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     2821 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     2881 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     2941 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     3001 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     3061 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     3121 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     3181 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     3241 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     3301 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     3361 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     3421 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3601 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
