LOCUS       sequence-238278-        4715 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 4715)
  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 4715)
  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-gal4-VP16-B-actin
FEATURES             Location/Qualifiers
     source          1..4715
                     /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..1102
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSF"
     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..1541
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     promoter        complement(2957..2975)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2980..2996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3098..3117)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3109..3918
                     /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(3176..3195)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4065..4653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4554..4573
                     /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 cggtccttct agaaagctac tgtcttctat cgaacaagca tgcgatattt
     1141 gccgacttaa aaagctcaag tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga
     1201 acaactggga gtgtcgctac tctcccaaaa ccaaaaggtc tccgctgact agggcacatc
     1261 tgacagaagt ggaatcaagg ctagaaagac tggaacagct atttctactg atttttcctc
     1321 gagaagacct tgacatgatt ttgaaaatgg attctttaca ggatataaaa gcattgttaa
     1381 caggattatt tgtacaagat aatgtgaata aagatgccgt cacagataga ttggcttcag
     1441 tggagactga tatgcctcta acattgagac agcatagaat aagtgcgaca tcatcatcgg
     1501 aagagagtag taacaaaggt caaagacagt tgactgtatc gtcgaggtcg accccgggaa
     1561 tacagatctc tcgagccgcc cccccgaccg atgtcagcct gggggacgag ctccacttag
     1621 acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt
     1681 tgggggacgg ggattccccg ggtccgggat ttacccccca cgactccgcc ccctacggcg
     1741 ctctggatat gtaaatcgat acggactgtt accacttcac gccgactcaa actgcgcaga
     1801 gaaacttcaa acgacaacat tggcatggct tttgttattt ttggcgcttg actcaggatc
     1861 taaaaactgg aacggtgaag gtgacggcaa tgttttttgg caaataagca tccccgaagt
     1921 tctacaatgc atctgaggac tccatgtttt gttttcttta gtcattccaa atgtttgtta
     1981 aatgcattgt tccgaaactt atttgcctct atgaaggctg cccagtaatt gggagcatac
     2041 ttaacattgt agtattgtat gtaaattatg taacaaaaca atgtctgggt ttttgtactt
     2101 ttcagcctta aaatcttggg tttatttttt tttctttgtt ccaaaaaaac gaagctttac
     2161 cattcaagat gtaaaggttt ccattccccc tgggcatatt gtaaaagctg tgtggaacgt
     2221 ggcggtgcca gacatttggt ggggccaacc tgtacactga ctaattcaat tccaataaaa
     2281 gtgcacatgt aagacatcct acccctgtct catttcctgt cttggctctg agtggcttca
     2341 ataaaaatgg cttttgtgaa gttgcaccat gttaattaca tttcagcact ttcacaccag
     2401 gtttttgact aatttgaatt tatcgaagtt tgaccttttt tttttttttt cttcaagcta
     2461 atttgaggtt atcaaaccat ttcattggag gaaacttaat gtttaaaagc ataaattcga
     2521 aacatgattc atttatttaa ctgcttaatg gtgaaacaaa cttcaagtaa tacaccaagt
     2581 gttccctcat atgaaggtag ctaactgcaa acggccttaa ctttccatct taagttgagc
     2641 tacataaagg ctttaacagg aaggaactgc cagtttaaaa gtcaaaccac catgactgat
     2701 ataaattctt gtccatataa ccttgcgttt cacacaatat agctatctac tgctaaataa
     2761 attaccatgg cgttgtctag caaggaagtg aagagctatc cggatatata tgtatgttat
     2821 cataactgta ctggctatgt caggtggtaa tgactgtaac gttacgttac tcgttgtagg
     2881 cacgacattg aatatgctct tctccgctgt ggcccgaacg cctcccgcta gcgatgggat
     2941 ctcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     3001 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     3061 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     3121 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     3181 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     3241 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3301 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3361 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     3421 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3481 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3541 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     3601 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3661 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     3721 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     3781 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     3841 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     3901 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     3961 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg
     4021 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4081 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4141 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4201 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4261 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4321 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4381 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4441 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4501 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4561 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4621 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4681 ttcctggcct tttgctggcc ttttgctcac atgtt
//
