LOCUS       sequence-218830-        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     137..156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(375..479)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      505..960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(592..611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     802..823
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1087..1109
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1101..1118
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1102..1118
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1125..1144
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1125..1143
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1176..1192
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1176..1192
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(2177..2179,2180..2182,2183..2893)
                     /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(2222..2243)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2483..2502)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2830..2851
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4184..4202)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4223..4239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4223..4239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4236..4258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4247..4263
                     /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(4271..4277,4278..4295,4296..4301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4316..4337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4454..4471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4625..5213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4705..4724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5384..5870,1..305,306..374))
                     /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"
ORIGIN
        1 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
       61 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      121 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      181 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      241 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      301 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      361 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      421 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      481 acatttcccc gaaaagtgcc acctacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      541 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      601 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      661 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      721 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      781 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
      841 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
      901 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
      961 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     1021 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     1081 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     1141 agggcgaatt ggagctccac cgcggtggcg gccgctctag aactagtgga tccggcggcg
     1201 ccctataaaa cccagcggcg cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag
     1261 cacagagcct cgcctttgcc gatccgccgc ccgtccacac ccgccgccag gtaagcccgg
     1321 ccagccgacc ggggcaggcg gctcacggcc cggccgcagg cggccgcggc cccttcgccc
     1381 gtgcagagcc gccgtctggg ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt
     1441 ggggggcgcg ggagaagccc ctgggcctcc ggagatgggg gacaccccac gccagttcgg
     1501 aggcgcgagg ccgcgctcgg gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa
     1561 ccggcggggt ctttgtctga gccgggctct tgccaatggg gatcgcaggg tgggcgcggc
     1621 ggagcccccg ccaggcccgg tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt
     1681 gggcgctaac tgcgtgcgcg ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca
     1741 aggcgctaac tgcgcgtgcg ttctggggcc cggggtgccg cggcctgggc tggggcgaag
     1801 gcgggctcgg ccggaagggg tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct
     1861 gcccgagccg ctggccgccc gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc
     1921 tgtttgaacc gggcggaggc ggggctggcg cccggttggg agggggttgg ggcctggctt
     1981 cctgccgcgc gccgcgggga cgcctccgac cagtgtttgc cttttatggt aataacgcgg
     2041 ccggcccggc ttcctttgtc cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag
     2101 gactcggcgc gccggaagtg gccagggcgg gggcgacctc ggctcacagc gcgcccggct
     2161 attctcgcaa ctgacaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2221 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2281 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2341 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2401 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2461 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2521 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2581 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2641 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2701 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2761 gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa
     2821 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2881 cgagctgtac aaggccggct ccggtaccga tgatgatatc gcagcgctcg tcgtcgacaa
     2941 cggctccggc atgtgcaagg ccggcttcgc gggcgacgat gccccccggg ccgtcttccc
     3001 ctccatcgtg gggcgcccca ggcaccaggt aggggagctg gctgggtggg gcagccccgg
     3061 gagcgggcgg gaggcaaggg cgctttctct gcacaggagc ctcccggttt ccggggtggg
     3121 ggctgcgccc gtgctcaggg cttcttgtcc tttccttccc agggcgtgat ggtgggcatg
     3181 ggtcagaagg attcctatgt gggcgacgag gcccagagca agagaggcat cctcaccctg
     3241 aagtacccca tcgagcacgg catcgtcacc aactgggacg acatggagaa aatctggcac
     3301 cacaccttct acaatgagct gcgtgtggct cccgaggagc accccgtgct gctgaccgag
     3361 gcccccctga accccaaggc caaccgcgag aagatgaccc aggtgagtgg cccgctacct
     3421 cttctggtgg ccgcctccct ccttcctggc ctcccggagc tgcgcccttt ctcactggtt
     3481 ctctcttctg ccgttttccg taggactctc ttctctgacc tgagtctcct ttggaactct
     3541 gcaggttcta tttgcttttt cccagatgag ctctttttct ggtgtttgtc tctctgacta
     3601 ggtgtctaag acagtgttgt gggtgtaggt actaacactg gctcgtgtga caaggccatg
     3661 aggctggtgt aaagcggcct tggagtgtgt attaagtagg cgcacagtag gtctgaacag
     3721 actccccatc ccaagacccc agcacactta gccgtgttct ttgcactttc tgcatgtccc
     3781 ccgtctggcc tggctgtccc cagtggcttc cccagtgtga catggtgtat ctctgcctta
     3841 cagatcatgt ttgagacctt caacacccca gccatgtacg ttgctatcca ggctgtgcta
     3901 tccctttgag gaagatgaac tgagatgtgt cagggtgact tatttccatc atcgtcctta
     3961 ggggagcttg ggtaggggca aggcgtgtag ctgggaccta ggtccagacc cctggctctg
     4021 ccactgaacg gctcaggtgc tttgggcagt tactccccgg cctcactttg cacgtgtgct
     4081 tacctagtgg agacaaaagt acatacctcg gtagagcgcg cacgcctgta accccaggtt
     4141 aactctctaa cctcgagggg gggcccggta ccagcttttg ttccctttag tgagggttaa
     4201 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4261 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4321 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4381 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4441 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5041 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5161 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5221 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5281 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5341 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5401 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5461 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5521 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5581 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5641 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5701 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5761 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5821 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
//
