LOCUS       sequence-223507-        5933 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 5933)
  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 5933)
  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..5933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..134
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     126..143
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     127..143
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     150..169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        150..168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     201..217
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     201..217
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1264..1266,1267..1980)
                     /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(1306..1327)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1567..1586)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1914..1935
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3272..3290)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3311..3327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3311..3327)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3324..3346)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3335..3351
                     /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(3359..3365,3366..3383,3384..3389))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3404..3425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3542..3559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3713..4301)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3793..3812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4472..5263,5264..5332))
                     /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     5095..5114
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5333..5437)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5463..5918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5550..5569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5760..5781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
       61 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
      121 gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc
      181 tccaccgcgg tggcggccgc tctagaacta gtggatccga cgcgtattgg gatgatgata
      241 ggaagtattt acagaacttt atagttagta actgactggt taatttttca aactgatttt
      301 tactcaactg aattagaaaa ggactggaaa gaaagtaaag atcccaacga cttgagggaa
      361 caagttggac aaccaaggac tttgtctaaa ttgtttttat ttagactaat gtggttctag
      421 ttctagagga ttcatactgg aatcatcggt ttaatattac gctatttgaa aggcagcata
      481 gtatagtact tttggaaaat tggcctgagg gtgatgtctt ttggaatatt tgtgaattca
      541 ctatgaagcc taattcctta aaaatgacct ccttcactca attatcagtg ttcttggttt
      601 gcctgggagt gaaaagagat cttaaaatct ttttggtttt agttacataa ttgactgatg
      661 taatattatg taatgatggc tgtacacagt gtctcatgcc ctataatcct agcactcatt
      721 tgagctcagg agttcaaggc cagcctgggc aacatggtaa aaccctgtct gcaccaaaaa
      781 taaaaaaaaa attagccggg catggtggca tgagtctgtg gttccagcta ctcaggaggc
      841 tgaggtgggg gaggatcgct tgagcccagg agtcagaggt tgcagtgagc tgagattgtg
      901 ctactgcact ccagcctggg ttacagagac cccatctgaa ttaaaaacat atataatgta
      961 atgatctgcc tcctttgtta acttgacttt tgaaatggga ttgtcagtag tatgatcatt
     1021 gttttcttgg atgccgactg tgtgtaaagt gttacatttt gaattaaatg tcagaatggg
     1081 tgaactttac taagattcaa ttctttgaat acaaagagca ttttattttg aagttagaat
     1141 actaattaaa tgcttatgac actttaaaaa attatttttt ttttctttca gagaattgta
     1201 agtgcacagt ccttggctga agatgatgtg gaaggcggta gcggggatcc accggtcgcc
     1261 accgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1321 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1381 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1441 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1501 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1561 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1621 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1681 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1741 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1801 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1861 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1921 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1981 tgagaaacaa atgtcaacat aataaaatct cagttaaaaa tattttaaaa attcttggta
     2041 gttgagcagc tctggggtaa taagggcaaa tatgcttgtt atgaactaca ctgaaatcta
     2101 ccaaagttaa tgtttacttt gtgtagatcc atttgtctat tttatttatt tttcccagtg
     2161 aaaagtgtat tttgatagag aacttttcat tctataaata cactatgagt tactaaaata
     2221 tcatggattt tgtttattcc tgaaacatag ttacatagtt aaactgtaca tatgacatgg
     2281 cttatgttaa aaatacccag tgctcagttt tgaaagatag gcaaaaaaaa aaagtatagg
     2341 agaaactgaa gaatgtacac ttttttagag ggcacatttt gctgtaaatc tggaaatttg
     2401 atagacttga ctgtgtttgt gaaaactgag cattaaaggt tttgattgat cctttctttc
     2461 catttaatct ctgagacgta aatatgtgag gtgtgctgct gtgctgggtt aacagcttcc
     2521 ttccctttct gtgtagcagt cttgaaatgt tctgtttaaa tcagtaggct taatgtgttc
     2581 tgggtattta tctccttgta ttttaaatat atgtagttgc aaatagcacc aggaattaga
     2641 tttctgtaca cccctaatct agccttgtga gcttcgctag ttaatgtgtg ctcactttcc
     2701 ctccatttgt tacgtgagag aatgcgtctg ctgatcactg aagtgtccct tttagcttct
     2761 gattcattgg gttctgttgg gcatctttaa atccacctta acctgaggaa tgtatgtggg
     2821 caaccaggcc ctgcattttt ttatattctg aattttgcat gcttgcctga cttagtattt
     2881 ctgaattgat gtttttttta atggtataac tatcttgatt ttcactgaaa ttatatggtt
     2941 ctgtcactac tctgtaaatt aatccgaaac ttttaaggta tcccaatggc gctttgagga
     3001 agatgaactg agatgtgtca gggtgactta tttccatcat cgtccttagg ggagcttggg
     3061 taggggcaag gcgtgtagct gggacctagg tccagacccc tggctctgcc actgaacggc
     3121 tcaggtgctt tgggcagtta ctccccggcc tcactttgca cgtgtgctta cctagtggag
     3181 acaaaagtac atacctcggt agagcgcgca cgcctgtaac cccaggttaa ctctctaacc
     3241 tcgagggggg gcccggtacc agcttttgtt ccctttagtg agggttaatt tcgagcttgg
     3301 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3361 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3421 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3481 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3541 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3601 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3661 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3721 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3781 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3841 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3901 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3961 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4021 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4081 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4141 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4201 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4261 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4321 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4381 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4441 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4501 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4561 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4621 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4681 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4741 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4801 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4861 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4921 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4981 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5041 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5101 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5161 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5221 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5281 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5341 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5401 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5461 ctacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5881 taacgcgaat tttaacaaaa tattaacgct tacaatttcc cattcgccat tca
//
