LOCUS       Exported                3729 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    Control-cx43.4-48H-pGTag
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3729)
  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 3729)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3729
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      268..295
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      387..473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             676..732
                     /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             733..1443
                     /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(1522..1539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1544..1678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1568..1587)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1622..1641
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1741..1836)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1970..1989)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1971..1989)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1994..2010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1994..2010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2112..2131)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2123..2932
                     /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(2190..2209)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3079..3667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3568..3587
                     /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 cggcccgttt tcttacgcgg ttgttggatg aaatctccaa ccactccacc
      661 ttcgtgggat ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
      721 aaccccggtc ctatggtgtc taagggcgag gaactgatta aggagaacat gcacatgaag
      781 ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc
      841 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
      901 ttcgccttcg acatcctggc taccagcttc atgtacggca gcagaacctt catcaaccac
      961 acccagggca tccccgactt ctttaagcag tccttccctg agggcttcac atgggagaga
     1021 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
     1081 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg
     1141 atgcagaaga aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc
     1201 ggcctggaag gcagaagcga catggccctg aagctcgtgg gcgggggcca cctgatctgc
     1261 aacttcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc
     1321 tactatgtgg accacagact ggaaagaatc aaggaggccg acaaagagac ctacgtcgag
     1381 cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg gcacaaactt
     1441 aataagctga accctcctga tgagagtggc cccggctgca tgagctgcaa gtgtgtgctc
     1501 tcctgaggcg cgcctctaga actatagtga gtcgtattac gtagatccag acatgataag
     1561 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1621 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     1681 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggttttttc
     1741 caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt tgttgcaacg
     1801 aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggcg ttgtctagca
     1861 aggaagtggg caagatatgg ctcacgttat tcatcatctt ccgcattgtt ttgaaaaccg
     1921 ctgtggcccg aacgcctccc gctagcgatg ggatctcggc cgcgatatcc cctatagtga
     1981 gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg
     2041 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     2101 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     2161 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     2221 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     2281 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     2341 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     2401 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     2461 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     2521 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     2581 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     2641 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     2701 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     2761 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     2821 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     2881 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     2941 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     3001 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     3061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3181 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     3241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3721 tcacatgtt
//
