LOCUS       Exported                5373 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human TOMM20.
ACCESSION   .
VERSION     .
KEYWORDS    AICSDP-8:TOMM20-mEGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5373)
  AUTHORS   Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, 
            Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, 
            Rafelski SM, Gunawardane RN
  TITLE     Systematic gene tagging using CRISPR/Cas9 in human stem cells to 
            illuminate cell organization.
  JOURNAL   Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 
            10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.
  PUBMED    28814507
REFERENCE   2  (bases 1 to 5373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5373
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1446..2162
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1488..1509)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1749..1768)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2096..2117
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3197..3213)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3197..3213)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3210..3232)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3221..3237
                     /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)."
     promoter        complement(3245..3275)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3290..3311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3428..3445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3599..4187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3679..3698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4358..5173)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5081..5100
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5174..5278)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5346..5364
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatgatga
      421 taggaagtat ttacagaact ttatagttag taactgactg gttaattttt caaactgatt
      481 tttactcaac tgaattagaa aaggactgga aagaaagtaa agatcccaac gacttgaggg
      541 aacaagttgg acaaccaagg actttgtcta aattgttttt atttagacta atgtggttct
      601 agttctagag gattcatact ggaatcatcg gtttaatatt acgctatttg aaaggcagca
      661 tagtatagta cttttggaaa attggcctga gggtgatgtc ttttggaata tttgtgaatt
      721 cactatgaag cctaattcct taaaaatgac ctccttcact caattatcag tgttcttggt
      781 ttgcctggga gtgaaaagag atcttaaaat ctttttggtt ttagttacat aattgactga
      841 tgtaatatta tgtaatgatg gctgtacaca gtgtctcatg ccctataatc ctagcactca
      901 tttgagctca ggagttcaag gccagcctgg gcaacatggt aaaaccctgt ctgcaccaaa
      961 aataaaaaaa aaattagccg ggcatggtgg catgagtctg tggttccagc tactcaggag
     1021 gctgaggtgg gggaggatcg cttgagccca ggagtcagag gttgcagtga gctgagattg
     1081 tgctactgca ctccagcctg ggttacagag accccatctg aattaaaaac atatataatg
     1141 taatgatctg cctcctttgt taacttgact tttgaaatgg gattgtcagt agtatgatca
     1201 ttgttttctt ggatgccgac tgtgtgtaaa gtgttacatt ttgaattaaa tgtcagaatg
     1261 ggtgaacttt actaagattc aattctttga atacaaagag cattttattt tgaagttaga
     1321 atactaatta aatgcttatg acactttaaa aaattatttt ttttttcttt cagagaattg
     1381 taagtgcaca gtccttggct gaagatgatg tggaaggcgg tagcggggat ccaccggtcg
     1441 ccaccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1501 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1561 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1621 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1681 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1741 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1801 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1861 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1921 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1981 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2041 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     2101 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     2161 aatgagaaac aaatgtcaac ataataaaat ctcagttaaa aatattttaa aaattcttgg
     2221 tagttgagca gctctggggt aataagggca aatatgcttg ttatgaacta cactgaaatc
     2281 taccaaagtt aatgtttact ttgtgtagat ccatttgtct attttattta tttttcccag
     2341 tgaaaagtgt attttgatag agaacttttc attctataaa tacactatga gttactaaaa
     2401 tatcatggat tttgtttatt cctgaaacat agttacatag ttaaactgta catatgacat
     2461 ggcttatgtt aaaaataccc agtgctcagt tttgaaagat aggcaaaaaa aaaaaagtat
     2521 aggagaaact gaagaatgta cactttttta gagggcacat tttgctgtaa atctggaaat
     2581 ttgatagact tgactgtgtt tgtgaaaact gagcattaaa ggttttgatt gatcctttct
     2641 ttccatttaa tctctgagac gtaaatatgt gaggtgtgct gctgtgctgg gttaacagct
     2701 tccttccctt tctgtgtagc agtcttgaaa tgttctgttt aaatcagtag gcttaatgtg
     2761 ttctgggtat ttatctcctt gtattttaaa tatatgtagt tgcaaatagc accaggaatt
     2821 agatttctgt acacccctaa tctagccttg tgagcttcgc tagttaatgt gtgctcactt
     2881 tccctccatt tgttacgtga gagaatgcgt ctgctgatca ctgaagtgtc ccttttagct
     2941 tctgattcat tgggttctgt tgggcatctt taaatccacc ttaacctgag gaatgtatgt
     3001 gggcaaccag gccctgcatt tttttatatt ctgaattttg catgcttgcc tgacttagta
     3061 tttctgaatt gatgtttttt ttaatggtat aactatcttg attttcactg aaattatatg
     3121 gttctgtcac tactctgtaa attaatccga aacttttaag gtatcccaat ggcgcgccga
     3181 gcttggctcg agcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3241 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3301 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3361 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3421 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3481 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3541 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3601 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3661 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3721 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3781 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3841 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3901 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3961 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4021 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4081 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4141 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4201 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4261 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4321 caatctaaag tatatatgag taaacttggt ctgacagtta gaaaaactca tcgagcatca
     4381 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     4441 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     4501 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     4561 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     4621 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     4681 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     4741 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4801 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4861 ttttcccagg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4921 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     4981 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     5041 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     5101 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     5161 gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5281 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5341 ataaaaatag gcgtatcacg aggccctttc gtc
//
