LOCUS       sequence-218837-        5934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert EGFP at the TOMM20 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5934)
  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 5934)
  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"; 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-mEGFP
FEATURES             Location/Qualifiers
     source          1..5934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..24
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8..24
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1071..1073,1074..1787)
                     /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(1113..1134)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1374..1393)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1721..1742
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3080..3098)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3119..3135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3119..3135)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3132..3154)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3143..3159
                     /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(3167..3173,3174..3191,3192..3197))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3212..3233
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3350..3367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3521..4109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3601..3620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4280..5071,5072..5140))
                     /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     4903..4922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5141..5245)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5271..5726)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5408..5429)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5620..5639
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5853..5875
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5867..5884
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5868..5884
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5891..5910
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5891..5909
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cggccgctct agaactagtg gatccgacgc gtattgggat gatgatagga agtatttaca
       61 gaactttata gttagtaact gactggttaa tttttcaaac tgatttttac tcaactgaat
      121 tagaaaagga ctggaaagaa agtaaagatc ccaacgactt gagggaacaa gttggacaac
      181 caaggacttt gtctaaattg tttttattta gactaatgtg gttctagttc tagaggattc
      241 atactggaat catcggttta atattacgct atttgaaagg cagcatagta tagtactttt
      301 ggaaaattgg cctgagggtg atgtcttttg gaatatttgt gaattcacta tgaagcctaa
      361 ttccttaaaa atgacctcct tcactcaatt atcagtgttc ttggtttgcc tgggagtgaa
      421 aagagatctt aaaatctttt tggttttagt tacataattg actgatgtaa tattatgtaa
      481 tgatggctgt acacagtgtc tcatgcccta taatcctagc actcatttga gctcaggagt
      541 tcaaggccag cctgggcaac atggtaaaac cctgtctgca ccaaaaataa aaaaaaaatt
      601 agccgggcat ggtggcatga gtctgtggtt ccagctactc aggaggctga ggtgggggag
      661 gatcgcttga gcccaggagt cagaggttgc agtgagctga gattgtgcta ctgcactcca
      721 gcctgggtta cagagacccc atctgaatta aaaacatata taatgtaatg atctgcctcc
      781 tttgttaact tgacttttga aatgggattg tcagtagtat gatcattgtt ttcttggatg
      841 ccgactgtgt gtaaagtgtt acattttgaa ttaaatgtca gaatgggtga actttactaa
      901 gattcaattc tttgaataca aagagcattt tattttgaag ttagaatact aattaaatgc
      961 ttatgacact ttaaaaaatt attttttttt tctttcagag aattgtaagt gcacagtcct
     1021 tggctgaaga tgatgtggaa ggcggtagcg gggatccacc ggtcgccacc gtgagcaagg
     1081 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1141 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1201 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1261 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1321 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1381 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1441 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1501 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1561 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1621 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1681 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1741 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatga gaaacaaatg
     1801 tcaacataat aaaatctcag ttaaaaatat tttaaaaatt cttggtagtt gagcagctct
     1861 ggggtaataa gggcaaatat gcttgttatg aactacactg aaatctacca aagttaatgt
     1921 ttactttgtg tagatccatt tgtctatttt atttattttt cccagtgaaa agtgtatttt
     1981 gatagagaac ttttcattct ataaatacac tatgagttac taaaatatca tggattttgt
     2041 ttattcctga aacatagtta catagttaaa ctgtacatat gacatggctt atgttaaaaa
     2101 tacccagtgc tcagttttga aagataggca aaaaaaaaaa agtataggag aaactgaaga
     2161 atgtacactt ttttagaggg cacattttgc tgtaaatctg gaaatttgat agacttgact
     2221 gtgtttgtga aaactgagca ttaaaggttt tgattgatcc tttctttcca tttaatctct
     2281 gagacgtaaa tatgtgaggt gtgctgctgt gctgggttaa cagcttcctt ccctttctgt
     2341 gtagcagtct tgaaatgttc tgtttaaatc agtaggctta atgtgttctg ggtatttatc
     2401 tccttgtatt ttaaatatat gtagttgcaa atagcaccag gaattagatt tctgtacacc
     2461 cctaatctag ccttgtgagc ttcgctagtt aatgtgtgct cactttccct ccatttgtta
     2521 cgtgagagaa tgcgtctgct gatcactgaa gtgtcccttt tagcttctga ttcattgggt
     2581 tctgttgggc atctttaaat ccaccttaac ctgaggaatg tatgtgggca accaggccct
     2641 gcattttttt atattctgaa ttttgcatgc ttgcctgact tagtatttct gaattgatgt
     2701 tttttttaat ggtataacta tcttgatttt cactgaaatt atatggttct gtcactactc
     2761 tgtaaattaa tccgaaactt ttaaggtatc ccaatggcgc tttgaggaag atgaactgag
     2821 atgtgtcagg gtgacttatt tccatcatcg tccttagggg agcttgggta ggggcaaggc
     2881 gtgtagctgg gacctaggtc cagacccctg gctctgccac tgaacggctc aggtgctttg
     2941 ggcagttact ccccggcctc actttgcacg tgtgcttacc tagtggagac aaaagtacat
     3001 acctcggtag agcgcgcacg cctgtaaccc caggttaact ctctaacctc gagggggggc
     3061 ccggtaccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt
     3121 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3181 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3241 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3301 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3361 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3421 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3481 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3541 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3601 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3661 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3721 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3781 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3841 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3901 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3961 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4021 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4081 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4141 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4201 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4261 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4321 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4381 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4441 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4501 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4561 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4621 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4681 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4741 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4801 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4861 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4921 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4981 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5041 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5101 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5161 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5221 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     5281 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5341 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5401 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5461 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5521 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5581 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5641 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5701 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     5761 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     5821 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     5881 cagtgaattg taatacgact cactataggg cgaattggag ctccaccgcg gtgg
//
