LOCUS       sequence-227719-        4816 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 4816)
  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 4816)
  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-B-actin
FEATURES             Location/Qualifiers
     source          1..4816
                     /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..1119
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSYGSKEEA"
     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             1103..1123
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1124..1126,1127..1129,1130..1843)
                     /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(1124..1126,1127..1129,1130..1840)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1169..1190)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1430..1449)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1777..1798
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3057..3075)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3080..3096)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3198..3217)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3209..4018
                     /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(3276..3295)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4165..4753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4654..4673
                     /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 ctccaaagaa gaagcgtaag gtaatggtga gcaagggcga
     1141 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1201 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1261 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1321 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1381 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1441 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1501 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1561 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1621 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1681 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1741 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1801 cgccgccggg atcactctcg gcatggacga gctgtacaag taactcgagg gcgcgccacg
     1861 gactgttacc acttcacgcc gactcaaact gcgcagagaa acttcaaacg acaacattgg
     1921 catggctttt gttatttttg gcgcttgact caggatctaa aaactggaac ggtgaaggtg
     1981 acggcaatgt tttttggcaa ataagcatcc ccgaagttct acaatgcatc tgaggactcc
     2041 atgttttgtt ttctttagtc attccaaatg tttgttaaat gcattgttcc gaaacttatt
     2101 tgcctctatg aaggctgccc agtaattggg agcatactta acattgtagt attgtatgta
     2161 aattatgtaa caaaacaatg tctgggtttt tgtacttttc agccttaaaa tcttgggttt
     2221 attttttttt ctttgttcca aaaaaacgaa gctttaccat tcaagatgta aaggtttcca
     2281 ttccccctgg gcatattgta aaagctgtgt ggaacgtggc ggtgccagac atttggtggg
     2341 gccaacctgt acactgacta attcaattcc aataaaagtg cacatgtaag acatcctacc
     2401 cctgtctcat ttcctgtctt ggctctgagt ggcttcaata aaaatggctt ttgtgaagtt
     2461 gcaccatgtt aattacattt cagcactttc acaccaggtt tttgactaat ttgaatttat
     2521 cgaagtttga cctttttttt tttttttttt cttcaagcta atttgaggtt atcaaaccat
     2581 ttcattggag gaaacttaat gtttaaaagc ataaattcga aacatgattc atttatttaa
     2641 ctgcttaatg gtgaaacaaa cttcaagtaa tacaccaagt gttccctcat atgaaggtag
     2701 ctaactgcaa acggccttaa ctttccatct taagttgagc tacataaagg ctttaacagg
     2761 aaggaactgc cagtttaaaa gtcaaaccac catgactgat ataaattctt gtccatataa
     2821 ccttgcgttt cacacaatat agctatctac tgctaaataa attaccatgg cgttgtctag
     2881 caaggaagtg aagagctatc cggatatata tgtatgttat cataactgta ctggctatgt
     2941 caggtggtaa tgactgtaac gttacgttac tcgttgtagg cacgacattg aatatgctct
     3001 tctccgctgt ggcccgaacg cctcccgcta gcgatgggat ctcggccgcg atatccccta
     3061 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     3121 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     3181 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     3241 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     3301 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     3361 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     3421 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     3481 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     3541 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     3601 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     3661 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     3721 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     3781 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     3841 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     3901 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     3961 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     4021 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc
     4081 aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     4141 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4201 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4261 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4321 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4381 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4441 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4501 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4561 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4621 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4681 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4741 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4801 ttttgctcac atgttc
//
