LOCUS       sequence-228477-        5870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert EGFP at the ACTB locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  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 5870)
  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(+)-ACTB-mEGFP
FEATURES             Location/Qualifiers
     source          1..5870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..64
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     56..73
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     57..73
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     80..99
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        80..98
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     131..147
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     131..147
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1132..1134,1135..1137,1138..1848)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1177..1198)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1438..1457)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1785..1806
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3139..3157)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3178..3194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3178..3194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3191..3213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3202..3218
                     /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(3226..3232,3233..3250,3251..3256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3271..3292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3409..3426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3580..4168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3660..3679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4339..5130,5131..5199))
                     /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     4962..4981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5200..5304)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5330..5785)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5467..5488)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5679..5698
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
       61 aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg
      121 tggcggccgc tctagaacta gtggatccgg cggcgcccta taaaacccag cggcgcgacg
      181 cgccaccacc gccgagaccg cgtccgcccc gcgagcacag agcctcgcct ttgccgatcc
      241 gccgcccgtc cacacccgcc gccaggtaag cccggccagc cgaccggggc aggcggctca
      301 cggcccggcc gcaggcggcc gcggcccctt cgcccgtgca gagccgccgt ctgggccgca
      361 gcggggggcg catggggggg gaaccggacc gccgtggggg gcgcgggaga agcccctggg
      421 cctccggaga tgggggacac cccacgccag ttcggaggcg cgaggccgcg ctcgggaggc
      481 gcgctccggg ggtgccgctc tcggggcggg ggcaaccggc ggggtctttg tctgagccgg
      541 gctcttgcca atggggatcg cagggtgggc gcggcggagc ccccgccagg cccggtgggg
      601 gctggggcgc cattgcgcgt gcgcgctggt cctttgggcg ctaactgcgt gcgcgctggg
      661 aattggcgct aattgcgcgt gcgcgctggg actcaaggcg ctaactgcgc gtgcgttctg
      721 gggcccgggg tgccgcggcc tgggctgggg cgaaggcggg ctcggccgga aggggtgggg
      781 tcgccgcggc tcccgggcgc ttgcgcgcac ttcctgcccg agccgctggc cgcccgaggg
      841 tgtggccgct gcgtgcgcgc gcgccgaccc ggcgctgttt gaaccgggcg gaggcggggc
      901 tggcgcccgg ttgggagggg gttggggcct ggcttcctgc cgcgcgccgc ggggacgcct
      961 ccgaccagtg tttgcctttt atggtaataa cgcggccggc ccggcttcct ttgtccccaa
     1021 tctgggcgcg cgccggcgcc ccctggcggc ctaaggactc ggcgcgccgg aagtggccag
     1081 ggcgggggcg acctcggctc acagcgcgcc cggctattct cgcaactgac aatggtgagc
     1141 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1201 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1261 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1321 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1381 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1441 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1501 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1561 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1621 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1681 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1741 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1801 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggc cggctccggt
     1861 accgatgatg atatcgcagc gctcgtcgtc gacaacggct ccggcatgtg caaggccggc
     1921 ttcgcgggcg acgatgcccc ccgggccgtc ttcccctcca tcgtggggcg ccccaggcac
     1981 caggtagggg agctggctgg gtggggcagc cccgggagcg ggcgggaggc aagggcgctt
     2041 tctctgcaca ggagcctccc ggtttccggg gtgggggctg cgcccgtgct cagggcttct
     2101 tgtcctttcc ttcccagggc gtgatggtgg gcatgggtca gaaggattcc tatgtgggcg
     2161 acgaggccca gagcaagaga ggcatcctca ccctgaagta ccccatcgag cacggcatcg
     2221 tcaccaactg ggacgacatg gagaaaatct ggcaccacac cttctacaat gagctgcgtg
     2281 tggctcccga ggagcacccc gtgctgctga ccgaggcccc cctgaacccc aaggccaacc
     2341 gcgagaagat gacccaggtg agtggcccgc tacctcttct ggtggccgcc tccctccttc
     2401 ctggcctccc ggagctgcgc cctttctcac tggttctctc ttctgccgtt ttccgtagga
     2461 ctctcttctc tgacctgagt ctcctttgga actctgcagg ttctatttgc tttttcccag
     2521 atgagctctt tttctggtgt ttgtctctct gactaggtgt ctaagacagt gttgtgggtg
     2581 taggtactaa cactggctcg tgtgacaagg ccatgaggct ggtgtaaagc ggccttggag
     2641 tgtgtattaa gtaggcgcac agtaggtctg aacagactcc ccatcccaag accccagcac
     2701 acttagccgt gttctttgca ctttctgcat gtcccccgtc tggcctggct gtccccagtg
     2761 gcttccccag tgtgacatgg tgtatctctg ccttacagat catgtttgag accttcaaca
     2821 ccccagccat gtacgttgct atccaggctg tgctatccct ttgaggaaga tgaactgaga
     2881 tgtgtcaggg tgacttattt ccatcatcgt ccttagggga gcttgggtag gggcaaggcg
     2941 tgtagctggg acctaggtcc agacccctgg ctctgccact gaacggctca ggtgctttgg
     3001 gcagttactc cccggcctca ctttgcacgt gtgcttacct agtggagaca aaagtacata
     3061 cctcggtaga gcgcgcacgc ctgtaacccc aggttaactc tctaacctcg agggggggcc
     3121 cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt aatcatggtc
     3181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     5341 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5401 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5461 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5521 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5581 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5641 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5701 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5761 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     5821 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
//
