LOCUS       sequence-238274-        4786 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 4786)
  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 4786)
  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-B-actin
FEATURES             Location/Qualifiers
     source          1..4786
                     /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..1978
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSYGEQGRGAVHRGGAHPGRAGRRRKRPQVQRVRRGRGRCHLRQADPE
                     VHLHHRQAARALAHPRDHPDLRRAVLQPLPRPHEAARLLQVRHARRLRPGAHHLLQGRR
                     QLQDPRRGEVRGRHPGEPHRAEGHRLQGGRQHPGAQAGVQLQQPQRLYHGRQAEERHQG
                     ELQDPPQHRGRQRAARRPLPAEHPHRRRPRAAARQPLPEHPVRPEQRPQREARSHGPAG
                     VRDRRRDHSRHGRAVQVTRGRATDCYHFTPTQTAQRNFKRQHWHGFCYFWRLTQDLKTG
                     TVKVTAMFFGK"
     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             join(1098..1100,1101..1103,1104..1817)
                     /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(1098..1100,1101..1103,1104..1814)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1143..1164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1404..1423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1751..1772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3028..3046)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3051..3067)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3169..3188)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3180..3989
                     /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(3247..3266)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4136..4724
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4625..4644
                     /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 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1201 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1261 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1321 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1381 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1501 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1561 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1621 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1681 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1801 acgagctgta caagtaactc gagggcgcgc cacggactgt taccacttca cgccgactca
     1861 aactgcgcag agaaacttca aacgacaaca ttggcatggc ttttgttatt tttggcgctt
     1921 gactcaggat ctaaaaactg gaacggtgaa ggtgacggca atgttttttg gcaaataagc
     1981 atccccgaag ttctacaatg catctgagga ctccatgttt tgttttcttt agtcattcca
     2041 aatgtttgtt aaatgcattg ttccgaaact tatttgcctc tatgaaggct gcccagtaat
     2101 tgggagcata cttaacattg tagtattgta tgtaaattat gtaacaaaac aatgtctggg
     2161 tttttgtact tttcagcctt aaaatcttgg gtttattttt ttttctttgt tccaaaaaaa
     2221 cgaagcttta ccattcaaga tgtaaaggtt tccattcccc ctgggcatat tgtaaaagct
     2281 gtgtggaacg tggcggtgcc agacatttgg tggggccaac ctgtacactg actaattcaa
     2341 ttccaataaa agtgcacatg taagacatcc tacccctgtc tcatttcctg tcttggctct
     2401 gagtggcttc aataaaaatg gcttttgtga agttgcacca tgttaattac atttcagcac
     2461 tttcacacca ggtttttgac taatttgaat ttatcgaagt ttgacctttt tttttttttt
     2521 tcttcaagct aatttgaggt tatcaaacca tttcattgga ggaaacttaa tgtttaaaag
     2581 cataaattcg aaacatgatt catttattta actgcttaat ggtgaaacaa acttcaagta
     2641 atacaccaag tgttccctca tatgaaggta gctaactgca aacggcctta actttccatc
     2701 ttaagttgag ctacataaag gctttaacag gaaggaactg ccagtttaaa agtcaaacca
     2761 ccatgactga tataaattct tgtccatata accttgcgtt tcacacaata tagctatcta
     2821 ctgctaaata aattaccatg gcgttgtcta gcaaggaagt gaagagctat ccggatatat
     2881 atgtatgtta tcataactgt actggctatg tcaggtggta atgactgtaa cgttacgtta
     2941 ctcgttgtag gcacgacatt gaatatgctc ttctccgctg tggcccgaac gcctcccgct
     3001 agcgatggga tctcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg
     3061 tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     3121 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     3181 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     3241 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     3301 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     3361 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     3421 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     3481 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     3541 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     3601 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     3661 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     3721 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     3781 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     3841 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     3901 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     3961 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc
     4021 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     4081 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4141 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4201 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4261 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4321 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4381 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4441 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4501 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4561 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4621 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4681 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4741 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
