LOCUS       sequence-223592-        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"
     primer_bind     46..68
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     60..77
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     61..77
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     84..103
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        84..102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     135..151
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     135..151
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1136..1867
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     promoter        complement(3164..3182)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3203..3219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3203..3219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3216..3238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3227..3243
                     /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(3251..3257,3258..3275,3276..3281))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3296..3317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3434..3451)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3605..4193)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3685..3704)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4364..5155,5156..5224))
                     /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     4987..5006
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5225..5329)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5355..5810)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5492..5513)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5704..5723
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
       61 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctccacc
      121 gcggtggcgg ccgctctaga actagtggat ccggcggcgc cctataaaac ccagcggcgc
      181 gacgcgccac caccgccgag accgcgtccg ccccgcgagc acagagcctc gcctttgccg
      241 atccgccgcc cgtccacacc cgccgccagg taagcccggc cagccgaccg gggcaggcgg
      301 ctcacggccc ggccgcaggc ggccgcggcc ccttcgcccg tgcagagccg ccgtctgggc
      361 cgcagcgggg ggcgcatggg gggggaaccg gaccgccgtg gggggcgcgg gagaagcccc
      421 tgggcctccg gagatggggg acaccccacg ccagttcgga ggcgcgaggc cgcgctcggg
      481 aggcgcgctc cgggggtgcc gctctcgggg cgggggcaac cggcggggtc tttgtctgag
      541 ccgggctctt gccaatgggg atcgcagggt gggcgcggcg gagcccccgc caggcccggt
      601 gggggctggg gcgccattgc gcgtgcgcgc tggtcctttg ggcgctaact gcgtgcgcgc
      661 tgggaattgg cgctaattgc gcgtgcgcgc tgggactcaa ggcgctaact gcgcgtgcgt
      721 tctggggccc ggggtgccgc ggcctgggct ggggcgaagg cgggctcggc cggaaggggt
      781 ggggtcgccg cggctcccgg gcgcttgcgc gcacttcctg cccgagccgc tggccgcccg
      841 agggtgtggc cgctgcgtgc gcgcgcgccg acccggcgct gtttgaaccg ggcggaggcg
      901 gggctggcgc ccggttggga gggggttggg gcctggcttc ctgccgcgcg ccgcggggac
      961 gcctccgacc agtgtttgcc ttttatggta ataacgcggc cggcccggct tcctttgtcc
     1021 ccaatctggg cgcgcgccgg cgccccctgg cggcctaagg actcggcgcg ccggaagtgg
     1081 ccagggcggg ggcgacctcg gctcacagcg cgcccggcta ttctcgcaac tgacaatggt
     1141 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     1201 cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     1261 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     1321 ggctaccagc ttcatgtacg gcagcagaac cttcatcaac cacacccagg gcatccccga
     1381 tttctttaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     1441 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     1501 cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
     1561 cggctgggag gccaacaccg agatgctgta ccccgctgac ggcggcctgg aaggcagaac
     1621 cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttca agaccacata
     1681 cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg tggaccacag
     1741 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt
     1801 ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaatggca tggacgagct
     1861 gtacaagtct agagccggct ccggtaccga tgatgatatc gcagcgctcg tcgtcgacaa
     1921 cggctccggc atgtgcaagg ccggcttcgc gggcgacgat gccccccggg ccgtcttccc
     1981 ctccatcgtg gggcgcccca ggcaccaggt aggggagctg gctgggtggg gcagccccgg
     2041 gagcgggcgg gaggcaaggg cgctttctct gcacaggagc ctcccggttt ccggggtggg
     2101 ggctgcgccc gtgctcaggg cttcttgtcc tttccttccc agggcgtgat ggtgggcatg
     2161 ggtcagaagg attcctatgt gggcgacgag gcccagagca agagaggcat cctcaccctg
     2221 aagtacccca tcgagcacgg catcgtcacc aactgggacg acatggagaa aatctggcac
     2281 cacaccttct acaatgagct gcgtgtggct cccgaggagc accccgtgct gctgaccgag
     2341 gcccccctga accccaaggc caaccgcgag aagatgaccc aggtgagtgg cccgctacct
     2401 cttctggtgg ccgcctccct ccttcctggc ctcccggagc tgcgcccttt ctcactggtt
     2461 ctctcttctg ccgttttccg taggactctc ttctctgacc tgagtctcct ttggaactct
     2521 gcaggttcta tttgcttttt cccagatgag ctctttttct ggtgtttgtc tctctgacta
     2581 ggtgtctaag acagtgttgt gggtgtaggt actaacactg gctcgtgtga caaggccatg
     2641 aggctggtgt aaagcggcct tggagtgtgt attaagtagg cgcacagtag gtctgaacag
     2701 actccccatc ccaagacccc agcacactta gccgtgttct ttgcactttc tgcatgtccc
     2761 ccgtctggcc tggctgtccc cagtggcttc cccagtgtga catggtgtat ctctgcctta
     2821 cagatcatgt ttgagacctt caacacccca gccatgtacg ttgctatcca ggctgtgcta
     2881 tccctttgag gaagatgaac tgagatgtgt cagggtgact tatttccatc atcgtcctta
     2941 ggggagcttg ggtaggggca aggcgtgtag ctgggaccta ggtccagacc cctggctctg
     3001 ccactgaacg gctcaggtgc tttgggcagt tactccccgg cctcactttg cacgtgtgct
     3061 tacctagtgg agacaaaagt acatacctcg gtagagcgcg cacgcctgta accccaggtt
     3121 aactctctaa cctcgagggg gggcccggta ccagcttttg ttccctttag tgagggttaa
     3181 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3241 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3301 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3361 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3421 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3481 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3541 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3601 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3661 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3721 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3781 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3841 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3901 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3961 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4021 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4081 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4141 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4201 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4261 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4321 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4381 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4441 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4501 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4561 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4621 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4681 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4741 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4801 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4861 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4921 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4981 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5041 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5101 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5161 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5221 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5281 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5341 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5401 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5461 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5521 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5581 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5641 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5701 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5761 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     5821 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     5881 gctggcgaaa g
//
