LOCUS       Exported                2958 bp ds-DNA     linear   SYN 02-JUN-2021
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    pGTag-TagRFP-B-actin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2958)
  AUTHORS   Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Weiss TJ, 
            Torrie ME, Vollbrecht MK, Lan M, McKeighan KC, Ming Z, Wehmeier A, 
            Mikelson CS, Haltom JA, Kwan KM, Chien CB, Balciunas D, Ekker SC, 
            Clark KJ, Webber BR, Moriarity B, Solin SL, Carlson DF, Dobbs DL, 
            McGrail M, Essner JJ.
  TITLE     GeneWeld: a method for efficient targeted integration directed by 
            short homology
  JOURNAL   bioRxiv 431627
REFERENCE   2  (bases 1 to 2958)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 2, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2958
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(169..187)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(192..208)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(310..329)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             321..1130
                     /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(388..407)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1277..1865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1766..1785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2019..2036
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      2195..2222
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2314..2400
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2464..2480
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2563..2584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2599..2629
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2637..2653
                     /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     2642..2664
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2661..2677
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2673..2957
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCH"
     primer_bind     2696..2716
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2698..2716
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(2764..2782)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2792..2808)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgaagagcta tccggatata tatgtatgtt atcataactg tactggctat gtcaggtggt
       61 aatgactgta acgttacgtt actcgttgta ggcacgacat tgaatatgct cttctccgct
      121 gtggcccgaa cgcctcccgc tagcgatggg atctcggccg cgatatcccc tatagtgagt
      181 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
      241 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
      301 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
      361 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
      421 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
      481 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
      541 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
      601 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
      661 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
      721 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
      781 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
      841 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
      901 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
      961 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     1021 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     1081 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     1141 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     1201 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     1261 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1321 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1381 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1441 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1501 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1561 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1621 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg
     1681 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1741 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1801 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1861 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1921 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1981 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2041 cggatgtgcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2101 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     2161 gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     2221 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     2281 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     2341 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     2401 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
     2461 gttgtaaaac gacggccagt cttaagctcg ggccctgcag ctctagagct cgaattcggg
     2521 aggcgttcgg gccacagcgg acacgcaggt gcgcaacgca attaatgtga gttagctcac
     2581 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     2641 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
     2701 taaccctcac taaagggaac aaaagctgga ggacctcgag ggggggcccg gtacccaatt
     2761 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     2821 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2881 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2941 gcgaatggac ctgccaca
//
