LOCUS       sequence-218829-        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"
     promoter        complement(280..298)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(319..335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(319..335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(332..354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    343..359
                     /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(367..373,374..391,392..397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    412..433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(550..567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(721..1309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(801..820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1480..2271,2272..2340))
                     /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     2103..2122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2341..2445)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2471..2926)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2608..2629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2820..2839
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3053..3075
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3067..3084
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3068..3084
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3091..3110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3091..3109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3142..3158
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3142..3158
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(4143..4145,4146..4148,4149..4859)
                     /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(4188..4209)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4449..4468)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4796..4817
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tttgaggaag atgaactgag atgtgtcagg gtgacttatt tccatcatcg tccttagggg
       61 agcttgggta ggggcaaggc gtgtagctgg gacctaggtc cagacccctg gctctgccac
      121 tgaacggctc aggtgctttg ggcagttact ccccggcctc actttgcacg tgtgcttacc
      181 tagtggagac aaaagtacat acctcggtag agcgcgcacg cctgtaaccc caggttaact
      241 ctctaacctc gagggggggc ccggtaccag cttttgttcc ctttagtgag ggttaatttc
      301 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      361 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      421 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      481 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      541 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      601 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      661 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      721 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      781 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      841 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      901 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      961 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1021 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1081 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1141 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1201 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1261 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1321 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1381 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1441 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1501 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1561 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1621 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1681 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1741 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1801 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1861 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1921 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1981 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2041 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2101 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2161 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2221 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2281 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2341 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2461 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     2521 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     2581 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     2641 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     2701 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     2761 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     2821 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2881 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
     2941 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     3001 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     3061 cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag
     3121 ctccaccgcg gtggcggccg ctctagaact agtggatccg gcggcgccct ataaaaccca
     3181 gcggcgcgac gcgccaccac cgccgagacc gcgtccgccc cgcgagcaca gagcctcgcc
     3241 tttgccgatc cgccgcccgt ccacacccgc cgccaggtaa gcccggccag ccgaccgggg
     3301 caggcggctc acggcccggc cgcaggcggc cgcggcccct tcgcccgtgc agagccgccg
     3361 tctgggccgc agcggggggc gcatgggggg ggaaccggac cgccgtgggg ggcgcgggag
     3421 aagcccctgg gcctccggag atgggggaca ccccacgcca gttcggaggc gcgaggccgc
     3481 gctcgggagg cgcgctccgg gggtgccgct ctcggggcgg gggcaaccgg cggggtcttt
     3541 gtctgagccg ggctcttgcc aatggggatc gcagggtggg cgcggcggag cccccgccag
     3601 gcccggtggg ggctggggcg ccattgcgcg tgcgcgctgg tcctttgggc gctaactgcg
     3661 tgcgcgctgg gaattggcgc taattgcgcg tgcgcgctgg gactcaaggc gctaactgcg
     3721 cgtgcgttct ggggcccggg gtgccgcggc ctgggctggg gcgaaggcgg gctcggccgg
     3781 aaggggtggg gtcgccgcgg ctcccgggcg cttgcgcgca cttcctgccc gagccgctgg
     3841 ccgcccgagg gtgtggccgc tgcgtgcgcg cgcgccgacc cggcgctgtt tgaaccgggc
     3901 ggaggcgggg ctggcgcccg gttgggaggg ggttggggcc tggcttcctg ccgcgcgccg
     3961 cggggacgcc tccgaccagt gtttgccttt tatggtaata acgcggccgg cccggcttcc
     4021 tttgtcccca atctgggcgc gcgccggcgc cccctggcgg cctaaggact cggcgcgccg
     4081 gaagtggcca gggcgggggc gacctcggct cacagcgcgc ccggctattc tcgcaactga
     4141 caatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     4201 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     4261 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     4321 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     4381 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     4441 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     4501 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     4561 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     4621 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     4681 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     4741 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     4801 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     4861 ccggctccgg taccgatgat gatatcgcag cgctcgtcgt cgacaacggc tccggcatgt
     4921 gcaaggccgg cttcgcgggc gacgatgccc cccgggccgt cttcccctcc atcgtggggc
     4981 gccccaggca ccaggtaggg gagctggctg ggtggggcag ccccgggagc gggcgggagg
     5041 caagggcgct ttctctgcac aggagcctcc cggtttccgg ggtgggggct gcgcccgtgc
     5101 tcagggcttc ttgtcctttc cttcccaggg cgtgatggtg ggcatgggtc agaaggattc
     5161 ctatgtgggc gacgaggccc agagcaagag aggcatcctc accctgaagt accccatcga
     5221 gcacggcatc gtcaccaact gggacgacat ggagaaaatc tggcaccaca ccttctacaa
     5281 tgagctgcgt gtggctcccg aggagcaccc cgtgctgctg accgaggccc ccctgaaccc
     5341 caaggccaac cgcgagaaga tgacccaggt gagtggcccg ctacctcttc tggtggccgc
     5401 ctccctcctt cctggcctcc cggagctgcg ccctttctca ctggttctct cttctgccgt
     5461 tttccgtagg actctcttct ctgacctgag tctcctttgg aactctgcag gttctatttg
     5521 ctttttccca gatgagctct ttttctggtg tttgtctctc tgactaggtg tctaagacag
     5581 tgttgtgggt gtaggtacta acactggctc gtgtgacaag gccatgaggc tggtgtaaag
     5641 cggccttgga gtgtgtatta agtaggcgca cagtaggtct gaacagactc cccatcccaa
     5701 gaccccagca cacttagccg tgttctttgc actttctgca tgtcccccgt ctggcctggc
     5761 tgtccccagt ggcttcccca gtgtgacatg gtgtatctct gccttacaga tcatgtttga
     5821 gaccttcaac accccagcca tgtacgttgc tatccaggct gtgctatccc
//
