LOCUS       sequence-223590-        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     49..71
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     63..80
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     64..80
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     87..106
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        87..105
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     138..154
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     138..154
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(1139..1141,1142..1144,1145..1855)
                     /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(1184..1205)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1445..1464)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1792..1813
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3146..3164)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3185..3201)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3185..3201)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3198..3220)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3209..3225
                     /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(3233..3239,3240..3257,3258..3263))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3278..3299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3416..3433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3587..4175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3667..3686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4346..5137,5138..5206))
                     /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     4969..4988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5207..5311)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5337..5792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5424..5443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5634..5655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
       61 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc
      121 accgcggtgg cggccgctct agaactagtg gatccggcgg cgccctataa aacccagcgg
      181 cgcgacgcgc caccaccgcc gagaccgcgt ccgccccgcg agcacagagc ctcgcctttg
      241 ccgatccgcc gcccgtccac acccgccgcc aggtaagccc ggccagccga ccggggcagg
      301 cggctcacgg cccggccgca ggcggccgcg gccccttcgc ccgtgcagag ccgccgtctg
      361 ggccgcagcg gggggcgcat ggggggggaa ccggaccgcc gtggggggcg cgggagaagc
      421 ccctgggcct ccggagatgg gggacacccc acgccagttc ggaggcgcga ggccgcgctc
      481 gggaggcgcg ctccgggggt gccgctctcg gggcgggggc aaccggcggg gtctttgtct
      541 gagccgggct cttgccaatg gggatcgcag ggtgggcgcg gcggagcccc cgccaggccc
      601 ggtgggggct ggggcgccat tgcgcgtgcg cgctggtcct ttgggcgcta actgcgtgcg
      661 cgctgggaat tggcgctaat tgcgcgtgcg cgctgggact caaggcgcta actgcgcgtg
      721 cgttctgggg cccggggtgc cgcggcctgg gctggggcga aggcgggctc ggccggaagg
      781 ggtggggtcg ccgcggctcc cgggcgcttg cgcgcacttc ctgcccgagc cgctggccgc
      841 ccgagggtgt ggccgctgcg tgcgcgcgcg ccgacccggc gctgtttgaa ccgggcggag
      901 gcggggctgg cgcccggttg ggagggggtt ggggcctggc ttcctgccgc gcgccgcggg
      961 gacgcctccg accagtgttt gccttttatg gtaataacgc ggccggcccg gcttcctttg
     1021 tccccaatct gggcgcgcgc cggcgccccc tggcggccta aggactcggc gcgccggaag
     1081 tggccagggc gggggcgacc tcggctcaca gcgcgcccgg ctattctcgc aactgacaat
     1141 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1201 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1261 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1321 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1381 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1441 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1501 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1561 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1621 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1681 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1741 cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1801 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggccgg
     1861 ctccggtacc gatgatgata tcgcagcgct cgtcgtcgac aacggctccg gcatgtgcaa
     1921 ggccggcttc gcgggcgacg atgccccccg ggccgtcttc ccctccatcg tggggcgccc
     1981 caggcaccag gtaggggagc tggctgggtg gggcagcccc gggagcgggc gggaggcaag
     2041 ggcgctttct ctgcacagga gcctcccggt ttccggggtg ggggctgcgc ccgtgctcag
     2101 ggcttcttgt cctttccttc ccagggcgtg atggtgggca tgggtcagaa ggattcctat
     2161 gtgggcgacg aggcccagag caagagaggc atcctcaccc tgaagtaccc catcgagcac
     2221 ggcatcgtca ccaactggga cgacatggag aaaatctggc accacacctt ctacaatgag
     2281 ctgcgtgtgg ctcccgagga gcaccccgtg ctgctgaccg aggcccccct gaaccccaag
     2341 gccaaccgcg agaagatgac ccaggtgagt ggcccgctac ctcttctggt ggccgcctcc
     2401 ctccttcctg gcctcccgga gctgcgccct ttctcactgg ttctctcttc tgccgttttc
     2461 cgtaggactc tcttctctga cctgagtctc ctttggaact ctgcaggttc tatttgcttt
     2521 ttcccagatg agctcttttt ctggtgtttg tctctctgac taggtgtcta agacagtgtt
     2581 gtgggtgtag gtactaacac tggctcgtgt gacaaggcca tgaggctggt gtaaagcggc
     2641 cttggagtgt gtattaagta ggcgcacagt aggtctgaac agactcccca tcccaagacc
     2701 ccagcacact tagccgtgtt ctttgcactt tctgcatgtc ccccgtctgg cctggctgtc
     2761 cccagtggct tccccagtgt gacatggtgt atctctgcct tacagatcat gtttgagacc
     2821 ttcaacaccc cagccatgta cgttgctatc caggctgtgc tatccctttg aggaagatga
     2881 actgagatgt gtcagggtga cttatttcca tcatcgtcct taggggagct tgggtagggg
     2941 caaggcgtgt agctgggacc taggtccaga cccctggctc tgccactgaa cggctcaggt
     3001 gctttgggca gttactcccc ggcctcactt tgcacgtgtg cttacctagt ggagacaaaa
     3061 gtacatacct cggtagagcg cgcacgcctg taaccccagg ttaactctct aacctcgagg
     3121 gggggcccgg taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     3181 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3241 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3301 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3361 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3421 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3481 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3541 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3601 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3661 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3721 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3781 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3841 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3901 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3961 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4021 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4081 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4141 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4201 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4261 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4321 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4381 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4441 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4501 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4561 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4621 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4681 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4741 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4801 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4861 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4921 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4981 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5041 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5101 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5161 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5221 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5281 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctacgc
     5341 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5401 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5461 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5521 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5581 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5641 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5701 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5761 gaattttaac aaaatattaa cgcttacaat ttcccattcg ccattcaggc tgcgcaactg
     5821 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
//
