LOCUS       sequence-227720-        4164 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 4164)
  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 4164)
  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-eGFP-CAAX-SV40
FEATURES             Location/Qualifiers
     source          1..4164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(267..294)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(386..472)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     536..552
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    635..656
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(671..676,677..694,695..701)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    709..725
                     /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     714..736
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     733..749
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             745..1830
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSYGEQGRGAVHRGGAHPGRAGRRRKRPQVQRVRRGRGRCHLRQADPE
                     VHLHHRQAARALAHPRDHPDLRRAVLQPLPRPHEAARLLQVRHARRLRPGAHHLLQGRR
                     QLQDPRRGEVRGRHPGEPHRAEGHRLQGGRQHPGAQAGVQLQQPQRLYHGRQAEERHQG
                     ELQDPPQHRGRQRAARRPLPAEHPHRRRPRAAARQPLPEHPVRPEQRPQREARSHGPAG
                     VRDRRRDHSRHGRAVQEAEPS"
     primer_bind     768..788
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        770..788
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(836..854)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(864..880)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1040..1096
                     /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             join(1097..1099,1100..1102,1103..1813)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1097..1099,1100..1102,1103..1813)
                     /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(1142..1163)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1403..1422)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1750..1771
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1914..2048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1938..1957)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1992..2011
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2111..2206)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2405..2423)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2428..2444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2546..2565)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2557..3366
                     /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(2624..2643)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3513..4101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4002..4021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
       61 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggatgtg
      121 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
      181 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct
      241 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
      301 tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca
      361 caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac
      421 aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg
      481 cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg acgttgtaaa
      541 acgacggcca gtcttaagct cgggccctgc agctctagag ctcgaattcg ggaggcgttc
      601 gggccacagc ggacacgcag gtgcgcaacg caattaatgt gagttagctc actcattagg
      661 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      721 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      781 actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa ttcgccctat
      841 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
      901 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      961 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1021 acctgccaca ggatccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga
     1081 agaaaacccc ggtcctatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1141 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1201 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1261 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1321 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1381 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1441 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1501 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1561 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1621 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1681 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1741 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1801 cgagctgtac aagaagctga accctcctga tgagagtggc cccggctgca tgagctgcaa
     1861 gtgtgtgctc tcctgaggcg cgcctctaga actatagtga gtcgtattac gtagatccag
     1921 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1981 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2041 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2101 aggttttttc caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt
     2161 tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggcg
     2221 ttgtctagca aggaagtgaa gagctatccg gatatatatg tatgttatca taactgtact
     2281 ggctatgtca ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa
     2341 tatgctcttc tccgctgtgg cccgaacgcc tcccgctagc gatgggatct cggccgcgat
     2401 atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg
     2461 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     2521 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     2581 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     2641 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     2701 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     2761 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     2821 cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta
     2881 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     2941 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     3001 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     3061 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     3121 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     3181 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     3241 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     3301 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     3361 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     3421 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     3481 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3541 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3601 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3661 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3721 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3781 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3841 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3901 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3961 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4021 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4081 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4141 tgctggcctt ttgctcacat gttc
//
