LOCUS       sequence-223532-        5918 bp    DNA     circular SYN 15-APR-2025
DEFINITION  donor to insert dTomato at the TOMM20 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  AUTHORS   Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J,
            Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, 
            Zamore PD, Sontheimer EJ, Wolfe SA
  TITLE     Efficient Homology-Directed Repair with Circular Single-Stranded DNA
            Donors.
  JOURNAL   CRISPR J. 2022 Oct;5(5):685-701. doi: 10.1089/crispr.2022.0058. Epub
            2022 Sep 7.
  PUBMED    36070530
REFERENCE   2  (bases 1 to 5918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1089/crispr.2022.0058"; journalName: "CRISPR J"; date: 
            "2022-10"; volume: "5"; issue: "5"; pages: "685-701"
COMMENT     Sequence Label: pBS-f1(+)-TOMM20-dTomato
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..130
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     122..139
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     123..139
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     146..165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        146..164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     197..213
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     197..213
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1260..1958
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLYGMDELYK"
     primer_bind     complement(1390..1408)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1658..1677
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1852..1875
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3253..3271)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3292..3308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3292..3308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3305..3327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3316..3332
                     /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(join(3340..3346,3347..3364,3365..3370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3385..3406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3523..3540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3694..4282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3774..3793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4453..5244,5245..5313))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5076..5095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5314..5418)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5444..5899)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5581..5602)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5793..5812
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
       61 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      121 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
      181 ccgcggtggc ggccgctcta gaactagtgg atccgacgcg tattgggatg atgataggaa
      241 gtatttacag aactttatag ttagtaactg actggttaat ttttcaaact gatttttact
      301 caactgaatt agaaaaggac tggaaagaaa gtaaagatcc caacgacttg agggaacaag
      361 ttggacaacc aaggactttg tctaaattgt ttttatttag actaatgtgg ttctagttct
      421 agaggattca tactggaatc atcggtttaa tattacgcta tttgaaaggc agcatagtat
      481 agtacttttg gaaaattggc ctgagggtga tgtcttttgg aatatttgtg aattcactat
      541 gaagcctaat tccttaaaaa tgacctcctt cactcaatta tcagtgttct tggtttgcct
      601 gggagtgaaa agagatctta aaatcttttt ggttttagtt acataattga ctgatgtaat
      661 attatgtaat gatggctgta cacagtgtct catgccctat aatcctagca ctcatttgag
      721 ctcaggagtt caaggccagc ctgggcaaca tggtaaaacc ctgtctgcac caaaaataaa
      781 aaaaaaatta gccgggcatg gtggcatgag tctgtggttc cagctactca ggaggctgag
      841 gtgggggagg atcgcttgag cccaggagtc agaggttgca gtgagctgag attgtgctac
      901 tgcactccag cctgggttac agagacccca tctgaattaa aaacatatat aatgtaatga
      961 tctgcctcct ttgttaactt gacttttgaa atgggattgt cagtagtatg atcattgttt
     1021 tcttggatgc cgactgtgtg taaagtgtta cattttgaat taaatgtcag aatgggtgaa
     1081 ctttactaag attcaattct ttgaatacaa agagcatttt attttgaagt tagaatacta
     1141 attaaatgct tatgacactt taaaaaatta tttttttttt ctttcagaga attgtaagtg
     1201 cacagtcctt ggctgaagat gatgtggaag gcggtagcgg ggatccaccg gtcgccaccg
     1261 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
     1321 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1381 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
     1441 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
     1501 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1561 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
     1621 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
     1681 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
     1741 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
     1801 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
     1861 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
     1921 accacctgtt cctgtacggc atggacgagc tgtacaagta atgagaaaca aatgtcaaca
     1981 taataaaatc tcagttaaaa atattttaaa aattcttggt agttgagcag ctctggggta
     2041 ataagggcaa atatgcttgt tatgaactac actgaaatct accaaagtta atgtttactt
     2101 tgtgtagatc catttgtcta ttttatttat ttttcccagt gaaaagtgta ttttgataga
     2161 gaacttttca ttctataaat acactatgag ttactaaaat atcatggatt ttgtttattc
     2221 ctgaaacata gttacatagt taaactgtac atatgacatg gcttatgtta aaaataccca
     2281 gtgctcagtt ttgaaagata ggcaaaaaaa aaaagtatag gagaaactga agaatgtaca
     2341 cttttttaga gggcacattt tgctgtaaat ctggaaattt gatagacttg actgtgtttg
     2401 tgaaaactga gcattaaagg ttttgattga tcctttcttt ccatttaatc tctgagacgt
     2461 aaatatgtga ggtgtgctgc tgtgctgggt taacagcttc cttccctttc tgtgtagcag
     2521 tcttgaaatg ttctgtttaa atcagtaggc ttaatgtgtt ctgggtattt atctccttgt
     2581 attttaaata tatgtagttg caaatagcac caggaattag atttctgtac acccctaatc
     2641 tagccttgtg agcttcgcta gttaatgtgt gctcactttc cctccatttg ttacgtgaga
     2701 gaatgcgtct gctgatcact gaagtgtccc ttttagcttc tgattcattg ggttctgttg
     2761 ggcatcttta aatccacctt aacctgagga atgtatgtgg gcaaccaggc cctgcatttt
     2821 tttatattct gaattttgca tgcttgcctg acttagtatt tctgaattga tgtttttttt
     2881 aatggtataa ctatcttgat tttcactgaa attatatggt tctgtcacta ctctgtaaat
     2941 taatccgaaa cttttaaggt atcccaatgg cgctttgagg aagatgaact gagatgtgtc
     3001 agggtgactt atttccatca tcgtccttag gggagcttgg gtaggggcaa ggcgtgtagc
     3061 tgggacctag gtccagaccc ctggctctgc cactgaacgg ctcaggtgct ttgggcagtt
     3121 actccccggc ctcactttgc acgtgtgctt acctagtgga gacaaaagta catacctcgg
     3181 tagagcgcgc acgcctgtaa ccccaggtta actctctaac ctcgaggggg ggcccggtac
     3241 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     3301 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3361 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3421 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3481 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3541 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3601 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3661 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3721 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3781 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3841 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3901 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3961 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4021 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4081 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4141 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4201 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4261 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4321 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4381 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4441 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4501 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4561 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4621 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4681 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4741 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4801 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4861 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4921 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4981 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5041 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5101 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5161 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5221 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5281 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5341 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5401 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5461 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5521 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5581 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5641 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5701 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5761 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5821 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5881 gcgccgctac agggcgcgtc ccattcgcca ttcaggct
//
