LOCUS       sequence-227741-        4774 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 4774)
  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 4774)
  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-TagRFP-B-actin
FEATURES             Location/Qualifiers
     source          1..4774
                     /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..1108
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHRIRSHELLS
                     VKASRRRGRKPRSYGV"
     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             1098..1811
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     promoter        complement(3016..3034)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3039..3055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3157..3176)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3168..3977
                     /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(3235..3254)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4124..4712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4613..4632
                     /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 gtgtctaagg gcgaggaact gattaaggag aacatgcaca
     1141 tgaagctgta catggagggc accgtgaaca accaccactt caagtgcaca tccgagggcg
     1201 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     1261 tccccttcgc cttcgacatc ctggctacca gcttcatgta cggcagcaga accttcatca
     1321 accacaccca gggcatcccc gacttcttta agcagtcctt ccctgagggc ttcacatggg
     1381 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     1441 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttccca tccaacggcc
     1501 ctgtgatgca gaagaaaaca ctcggctggg aggccaacac cgagatgctg taccccgctg
     1561 acggcggcct ggaaggcaga agcgacatgg ccctgaagct cgtgggcggg ggccacctga
     1621 tctgcaactt caagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg
     1681 gcgtctacta tgtggaccac agactggaaa gaatcaagga ggccgacaaa gagacctacg
     1741 tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1801 aacttaattg aggcgcgcca cggactgtta ccacttcacg ccgactcaaa ctgcgcagag
     1861 aaacttcaaa cgacaacatt ggcatggctt ttgttatttt tggcgcttga ctcaggatct
     1921 aaaaactgga acggtgaagg tgacggcaat gttttttggc aaataagcat ccccgaagtt
     1981 ctacaatgca tctgaggact ccatgttttg ttttctttag tcattccaaa tgtttgttaa
     2041 atgcattgtt ccgaaactta tttgcctcta tgaaggctgc ccagtaattg ggagcatact
     2101 taacattgta gtattgtatg taaattatgt aacaaaacaa tgtctgggtt tttgtacttt
     2161 tcagccttaa aatcttgggt ttattttttt ttctttgttc caaaaaaacg aagctttacc
     2221 attcaagatg taaaggtttc cattccccct gggcatattg taaaagctgt gtggaacgtg
     2281 gcggtgccag acatttggtg gggccaacct gtacactgac taattcaatt ccaataaaag
     2341 tgcacatgta agacatccta cccctgtctc atttcctgtc ttggctctga gtggcttcaa
     2401 taaaaatggc ttttgtgaag ttgcaccatg ttaattacat ttcagcactt tcacaccagg
     2461 tttttgacta atttgaattt atcgaagttt gacctttttt tttttttttc ttcaagctaa
     2521 tttgaggtta tcaaaccatt tcattggagg aaacttaatg tttaaaagca taaattcgaa
     2581 acatgattca tttatttaac tgcttaatgg tgaaacaaac ttcaagtaat acaccaagtg
     2641 ttccctcata tgaaggtagc taactgcaaa cggccttaac tttccatctt aagttgagct
     2701 acataaaggc tttaacagga aggaactgcc agtttaaaag tcaaaccacc atgactgata
     2761 taaattcttg tccatataac cttgcgtttc acacaatata gctatctact gctaaataaa
     2821 ttaccatggc gttgtctagc aaggaagtga agagctatcc ggatatatat gtatgttatc
     2881 ataactgtac tggctatgtc aggtggtaat gactgtaacg ttacgttact cgttgtaggc
     2941 acgacattga atatgctctt ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc
     3001 tcggccgcga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     3061 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     3121 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     3181 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3241 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     3301 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3361 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3421 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     3481 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3541 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3601 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3661 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     3721 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     3781 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3841 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3901 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     3961 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     4021 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt
     4081 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4141 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4201 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4261 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4321 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4381 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4441 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4501 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4561 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4621 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4681 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4741 tcctggcctt ttgctggcct tttgctcaca tgtt
//
