LOCUS       sequence-238276-        4840 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 4840)
  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 4840)
  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-B-actin
FEATURES             Location/Qualifiers
     source          1..4840
                     /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"
     promoter        complement(3081..3099)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3104..3120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3222..3241)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3233..4042
                     /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(3300..3319)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4189..4777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4678..4697
                     /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 cgccacggac tgttaccact tcacgccgac tcaaactgcg
     1921 cagagaaact tcaaacgaca acattggcat ggcttttgtt atttttggcg cttgactcag
     1981 gatctaaaaa ctggaacggt gaaggtgacg gcaatgtttt ttggcaaata agcatccccg
     2041 aagttctaca atgcatctga ggactccatg ttttgttttc tttagtcatt ccaaatgttt
     2101 gttaaatgca ttgttccgaa acttatttgc ctctatgaag gctgcccagt aattgggagc
     2161 atacttaaca ttgtagtatt gtatgtaaat tatgtaacaa aacaatgtct gggtttttgt
     2221 acttttcagc cttaaaatct tgggtttatt tttttttctt tgttccaaaa aaacgaagct
     2281 ttaccattca agatgtaaag gtttccattc cccctgggca tattgtaaaa gctgtgtgga
     2341 acgtggcggt gccagacatt tggtggggcc aacctgtaca ctgactaatt caattccaat
     2401 aaaagtgcac atgtaagaca tcctacccct gtctcatttc ctgtcttggc tctgagtggc
     2461 ttcaataaaa atggcttttg tgaagttgca ccatgttaat tacatttcag cactttcaca
     2521 ccaggttttt gactaatttg aatttatcga agtttgacct tttttttttt ttttcttcaa
     2581 gctaatttga ggttatcaaa ccatttcatt ggaggaaact taatgtttaa aagcataaat
     2641 tcgaaacatg attcatttat ttaactgctt aatggtgaaa caaacttcaa gtaatacacc
     2701 aagtgttccc tcatatgaag gtagctaact gcaaacggcc ttaactttcc atcttaagtt
     2761 gagctacata aaggctttaa caggaaggaa ctgccagttt aaaagtcaaa ccaccatgac
     2821 tgatataaat tcttgtccat ataaccttgc gtttcacaca atatagctat ctactgctaa
     2881 ataaattacc atggcgttgt ctagcaagga agtgaagagc tatccggata tatatgtatg
     2941 ttatcataac tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg
     3001 taggcacgac attgaatatg ctcttctccg ctgtggcccg aacgcctccc gctagcgatg
     3061 ggatctcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     3121 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     3181 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     3241 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     3301 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     3361 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3421 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3481 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     3541 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3601 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3661 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3721 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3781 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3841 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3901 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3961 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     4021 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     4081 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     4141 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4501 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4801 acggttcctg gccttttgct ggccttttgc tcacatgttc
//
