LOCUS       sequence-223506-        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     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     42..61
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        42..60
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     93..109
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     93..109
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1156..1158,1159..1872)
                     /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(1198..1219)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1459..1478)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1806..1827
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3165..3183)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3204..3220)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3204..3220)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3217..3239)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3228..3244
                     /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(3252..3258,3259..3276,3277..3282))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3297..3318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3435..3452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3606..4194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3686..3705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4365..5156,5157..5225))
                     /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     4988..5007
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5226..5330)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5356..5811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5493..5514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5705..5724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
       61 gcgaattgga gctccaccgc ggtggcggcc gctctagaac tagtggatcc gacgcgtatt
      121 gggatgatga taggaagtat ttacagaact ttatagttag taactgactg gttaattttt
      181 caaactgatt tttactcaac tgaattagaa aaggactgga aagaaagtaa agatcccaac
      241 gacttgaggg aacaagttgg acaaccaagg actttgtcta aattgttttt atttagacta
      301 atgtggttct agttctagag gattcatact ggaatcatcg gtttaatatt acgctatttg
      361 aaaggcagca tagtatagta cttttggaaa attggcctga gggtgatgtc ttttggaata
      421 tttgtgaatt cactatgaag cctaattcct taaaaatgac ctccttcact caattatcag
      481 tgttcttggt ttgcctggga gtgaaaagag atcttaaaat ctttttggtt ttagttacat
      541 aattgactga tgtaatatta tgtaatgatg gctgtacaca gtgtctcatg ccctataatc
      601 ctagcactca tttgagctca ggagttcaag gccagcctgg gcaacatggt aaaaccctgt
      661 ctgcaccaaa aataaaaaaa aaattagccg ggcatggtgg catgagtctg tggttccagc
      721 tactcaggag gctgaggtgg gggaggatcg cttgagccca ggagtcagag gttgcagtga
      781 gctgagattg tgctactgca ctccagcctg ggttacagag accccatctg aattaaaaac
      841 atatataatg taatgatctg cctcctttgt taacttgact tttgaaatgg gattgtcagt
      901 agtatgatca ttgttttctt ggatgccgac tgtgtgtaaa gtgttacatt ttgaattaaa
      961 tgtcagaatg ggtgaacttt actaagattc aattctttga atacaaagag cattttattt
     1021 tgaagttaga atactaatta aatgcttatg acactttaaa aaattatttt ttttttcttt
     1081 cagagaattg taagtgcaca gtccttggct gaagatgatg tggaaggcgg tagcggggat
     1141 ccaccggtcg ccaccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1201 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1261 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1321 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1381 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1441 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1501 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1561 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1621 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1681 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1741 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     1801 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1861 ctgtacaagt aatgagaaac aaatgtcaac ataataaaat ctcagttaaa aatattttaa
     1921 aaattcttgg tagttgagca gctctggggt aataagggca aatatgcttg ttatgaacta
     1981 cactgaaatc taccaaagtt aatgtttact ttgtgtagat ccatttgtct attttattta
     2041 tttttcccag tgaaaagtgt attttgatag agaacttttc attctataaa tacactatga
     2101 gttactaaaa tatcatggat tttgtttatt cctgaaacat agttacatag ttaaactgta
     2161 catatgacat ggcttatgtt aaaaataccc agtgctcagt tttgaaagat aggcaaaaaa
     2221 aaaaaagtat aggagaaact gaagaatgta cactttttta gagggcacat tttgctgtaa
     2281 atctggaaat ttgatagact tgactgtgtt tgtgaaaact gagcattaaa ggttttgatt
     2341 gatcctttct ttccatttaa tctctgagac gtaaatatgt gaggtgtgct gctgtgctgg
     2401 gttaacagct tccttccctt tctgtgtagc agtcttgaaa tgttctgttt aaatcagtag
     2461 gcttaatgtg ttctgggtat ttatctcctt gtattttaaa tatatgtagt tgcaaatagc
     2521 accaggaatt agatttctgt acacccctaa tctagccttg tgagcttcgc tagttaatgt
     2581 gtgctcactt tccctccatt tgttacgtga gagaatgcgt ctgctgatca ctgaagtgtc
     2641 ccttttagct tctgattcat tgggttctgt tgggcatctt taaatccacc ttaacctgag
     2701 gaatgtatgt gggcaaccag gccctgcatt tttttatatt ctgaattttg catgcttgcc
     2761 tgacttagta tttctgaatt gatgtttttt ttaatggtat aactatcttg attttcactg
     2821 aaattatatg gttctgtcac tactctgtaa attaatccga aacttttaag gtatcccaat
     2881 ggcgctttga ggaagatgaa ctgagatgtg tcagggtgac ttatttccat catcgtcctt
     2941 aggggagctt gggtaggggc aaggcgtgta gctgggacct aggtccagac ccctggctct
     3001 gccactgaac ggctcaggtg ctttgggcag ttactccccg gcctcacttt gcacgtgtgc
     3061 ttacctagtg gagacaaaag tacatacctc ggtagagcgc gcacgcctgt aaccccaggt
     3121 taactctcta acctcgaggg ggggcccggt accagctttt gttcccttta gtgagggtta
     3181 atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3241 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3301 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3361 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3421 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3481 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3541 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3601 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3661 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3721 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3781 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3841 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3901 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3961 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4021 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4081 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4141 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4201 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4261 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4321 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4381 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4441 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4501 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4561 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4621 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4681 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4741 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4801 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4861 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4921 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4981 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5041 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5101 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5161 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5221 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5281 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5341 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5401 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5461 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5521 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5581 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5641 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5701 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5761 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     5821 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5881 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggt
//
