LOCUS       sequence-218831-        5891 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert iTagRFP-T at the ACTB locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5891)
  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 5891)
  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-iTagRFP-T
FEATURES             Location/Qualifiers
     source          1..5891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(24..612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(104..123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(783..1574,1575..1643))
                     /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     1406..1425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1644..1748)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1774..2229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1861..1880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2071..2092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2356..2378
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2370..2387
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2371..2387
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2394..2413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2394..2412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2445..2461
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2445..2461
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3446..4177
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     promoter        complement(5474..5492)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5513..5529)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5513..5529)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5526..5548)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5537..5553
                     /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(5561..5567,5568..5585,5586..5591))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5606..5627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5744..5761)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
       61 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
      601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
      661 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      721 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      781 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      841 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      901 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
      961 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     1021 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     1081 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     1141 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     1201 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1261 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1321 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1381 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1441 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1501 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1561 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1621 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1681 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1741 gttccgcgca catttccccg aaaagtgcca cctacgcgcc ctgtagcggc gcattaagcg
     1801 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1861 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1921 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1981 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2041 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2101 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2221 ttacaatttc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     2281 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     2341 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     2401 actcactata gggcgaattg gagctccacc gcggtggcgg ccgctctaga actagtggat
     2461 ccggcggcgc cctataaaac ccagcggcgc gacgcgccac caccgccgag accgcgtccg
     2521 ccccgcgagc acagagcctc gcctttgccg atccgccgcc cgtccacacc cgccgccagg
     2581 taagcccggc cagccgaccg gggcaggcgg ctcacggccc ggccgcaggc ggccgcggcc
     2641 ccttcgcccg tgcagagccg ccgtctgggc cgcagcgggg ggcgcatggg gggggaaccg
     2701 gaccgccgtg gggggcgcgg gagaagcccc tgggcctccg gagatggggg acaccccacg
     2761 ccagttcgga ggcgcgaggc cgcgctcggg aggcgcgctc cgggggtgcc gctctcgggg
     2821 cgggggcaac cggcggggtc tttgtctgag ccgggctctt gccaatgggg atcgcagggt
     2881 gggcgcggcg gagcccccgc caggcccggt gggggctggg gcgccattgc gcgtgcgcgc
     2941 tggtcctttg ggcgctaact gcgtgcgcgc tgggaattgg cgctaattgc gcgtgcgcgc
     3001 tgggactcaa ggcgctaact gcgcgtgcgt tctggggccc ggggtgccgc ggcctgggct
     3061 ggggcgaagg cgggctcggc cggaaggggt ggggtcgccg cggctcccgg gcgcttgcgc
     3121 gcacttcctg cccgagccgc tggccgcccg agggtgtggc cgctgcgtgc gcgcgcgccg
     3181 acccggcgct gtttgaaccg ggcggaggcg gggctggcgc ccggttggga gggggttggg
     3241 gcctggcttc ctgccgcgcg ccgcggggac gcctccgacc agtgtttgcc ttttatggta
     3301 ataacgcggc cggcccggct tcctttgtcc ccaatctggg cgcgcgccgg cgccccctgg
     3361 cggcctaagg actcggcgcg ccggaagtgg ccagggcggg ggcgacctcg gctcacagcg
     3421 cgcccggcta ttctcgcaac tgacaatggt gtctaagggc gaagagctga ttaaggagaa
     3481 catgcacatg aagctgtaca tggagggcac cgtgaacaac caccacttca agtgcacatc
     3541 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     3601 cggccctctc cccttcgcct tcgacatcct ggctaccagc ttcatgtacg gcagcagaac
     3661 cttcatcaac cacacccagg gcatccccga tttctttaag cagtccttcc ctgagggctt
     3721 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     3781 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcccatc
     3841 caacggccct gtgatgcaga agaaaacact cggctgggag gccaacaccg agatgctgta
     3901 ccccgctgac ggcggcctgg aaggcagaac cgacatggcc ctgaagctcg tgggcggggg
     3961 ccacctgatc tgcaacttca agaccacata cagatccaag aaacccgcta agaacctcaa
     4021 gatgcccggc gtctactatg tggaccacag actggaaaga atcaaggagg ccgacaaaga
     4081 gacctacgtc gagcagcacg aggtggctgt ggccagatac tgcgacctcc ctagcaaact
     4141 ggggcacaaa cttaatggca tggacgagct gtacaagtct agagccggct ccggtaccga
     4201 tgatgatatc gcagcgctcg tcgtcgacaa cggctccggc atgtgcaagg ccggcttcgc
     4261 gggcgacgat gccccccggg ccgtcttccc ctccatcgtg gggcgcccca ggcaccaggt
     4321 aggggagctg gctgggtggg gcagccccgg gagcgggcgg gaggcaaggg cgctttctct
     4381 gcacaggagc ctcccggttt ccggggtggg ggctgcgccc gtgctcaggg cttcttgtcc
     4441 tttccttccc agggcgtgat ggtgggcatg ggtcagaagg attcctatgt gggcgacgag
     4501 gcccagagca agagaggcat cctcaccctg aagtacccca tcgagcacgg catcgtcacc
     4561 aactgggacg acatggagaa aatctggcac cacaccttct acaatgagct gcgtgtggct
     4621 cccgaggagc accccgtgct gctgaccgag gcccccctga accccaaggc caaccgcgag
     4681 aagatgaccc aggtgagtgg cccgctacct cttctggtgg ccgcctccct ccttcctggc
     4741 ctcccggagc tgcgcccttt ctcactggtt ctctcttctg ccgttttccg taggactctc
     4801 ttctctgacc tgagtctcct ttggaactct gcaggttcta tttgcttttt cccagatgag
     4861 ctctttttct ggtgtttgtc tctctgacta ggtgtctaag acagtgttgt gggtgtaggt
     4921 actaacactg gctcgtgtga caaggccatg aggctggtgt aaagcggcct tggagtgtgt
     4981 attaagtagg cgcacagtag gtctgaacag actccccatc ccaagacccc agcacactta
     5041 gccgtgttct ttgcactttc tgcatgtccc ccgtctggcc tggctgtccc cagtggcttc
     5101 cccagtgtga catggtgtat ctctgcctta cagatcatgt ttgagacctt caacacccca
     5161 gccatgtacg ttgctatcca ggctgtgcta tccctttgag gaagatgaac tgagatgtgt
     5221 cagggtgact tatttccatc atcgtcctta ggggagcttg ggtaggggca aggcgtgtag
     5281 ctgggaccta ggtccagacc cctggctctg ccactgaacg gctcaggtgc tttgggcagt
     5341 tactccccgg cctcactttg cacgtgtgct tacctagtgg agacaaaagt acatacctcg
     5401 gtagagcgcg cacgcctgta accccaggtt aactctctaa cctcgagggg gggcccggta
     5461 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     5521 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5581 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5641 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5701 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5761 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5821 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5881 ccaggaaccg t
//
