LOCUS       sequence-218832-        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"
     CDS             1..732
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     promoter        complement(2029..2047)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2068..2084)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2068..2084)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2081..2103)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2092..2108
                     /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(2116..2122,2123..2140,2141..2146))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2161..2182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2299..2316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2470..3058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2550..2569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3229..4020,4021..4089))
                     /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     3852..3871
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4090..4194)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4220..4675)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4357..4378)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4569..4588
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4802..4824
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4816..4833
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4817..4833
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4840..4859
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4840..4858
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4891..4907
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4891..4907
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag
       61 ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
      121 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
      181 atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac ccagggcatc
      241 cccgatttct ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
      301 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
      361 tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
      421 acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg cctggaaggc
      481 agaaccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttcaagacc
      541 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac
      601 cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg
      661 gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa tggcatggac
      721 gagctgtaca agtctagagc cggctccggt accgatgatg atatcgcagc gctcgtcgtc
      781 gacaacggct ccggcatgtg caaggccggc ttcgcgggcg acgatgcccc ccgggccgtc
      841 ttcccctcca tcgtggggcg ccccaggcac caggtagggg agctggctgg gtggggcagc
      901 cccgggagcg ggcgggaggc aagggcgctt tctctgcaca ggagcctccc ggtttccggg
      961 gtgggggctg cgcccgtgct cagggcttct tgtcctttcc ttcccagggc gtgatggtgg
     1021 gcatgggtca gaaggattcc tatgtgggcg acgaggccca gagcaagaga ggcatcctca
     1081 ccctgaagta ccccatcgag cacggcatcg tcaccaactg ggacgacatg gagaaaatct
     1141 ggcaccacac cttctacaat gagctgcgtg tggctcccga ggagcacccc gtgctgctga
     1201 ccgaggcccc cctgaacccc aaggccaacc gcgagaagat gacccaggtg agtggcccgc
     1261 tacctcttct ggtggccgcc tccctccttc ctggcctccc ggagctgcgc cctttctcac
     1321 tggttctctc ttctgccgtt ttccgtagga ctctcttctc tgacctgagt ctcctttgga
     1381 actctgcagg ttctatttgc tttttcccag atgagctctt tttctggtgt ttgtctctct
     1441 gactaggtgt ctaagacagt gttgtgggtg taggtactaa cactggctcg tgtgacaagg
     1501 ccatgaggct ggtgtaaagc ggccttggag tgtgtattaa gtaggcgcac agtaggtctg
     1561 aacagactcc ccatcccaag accccagcac acttagccgt gttctttgca ctttctgcat
     1621 gtcccccgtc tggcctggct gtccccagtg gcttccccag tgtgacatgg tgtatctctg
     1681 ccttacagat catgtttgag accttcaaca ccccagccat gtacgttgct atccaggctg
     1741 tgctatccct ttgaggaaga tgaactgaga tgtgtcaggg tgacttattt ccatcatcgt
     1801 ccttagggga gcttgggtag gggcaaggcg tgtagctggg acctaggtcc agacccctgg
     1861 ctctgccact gaacggctca ggtgctttgg gcagttactc cccggcctca ctttgcacgt
     1921 gtgcttacct agtggagaca aaagtacata cctcggtaga gcgcgcacgc ctgtaacccc
     1981 aggttaactc tctaacctcg agggggggcc cggtaccagc ttttgttccc tttagtgagg
     2041 gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2101 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2161 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2221 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2281 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2341 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2401 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2461 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2521 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2581 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2641 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2701 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2761 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2821 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2881 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2941 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3001 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3061 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3121 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3181 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3241 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3301 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3361 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3421 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3481 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3541 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3601 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3661 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3721 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3781 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3841 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3901 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3961 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4021 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4081 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4141 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4201 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4261 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4321 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4381 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4441 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4501 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4561 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4621 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     4681 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     4741 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     4801 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
     4861 gaattggagc tccaccgcgg tggcggccgc tctagaacta gtggatccgg cggcgcccta
     4921 taaaacccag cggcgcgacg cgccaccacc gccgagaccg cgtccgcccc gcgagcacag
     4981 agcctcgcct ttgccgatcc gccgcccgtc cacacccgcc gccaggtaag cccggccagc
     5041 cgaccggggc aggcggctca cggcccggcc gcaggcggcc gcggcccctt cgcccgtgca
     5101 gagccgccgt ctgggccgca gcggggggcg catggggggg gaaccggacc gccgtggggg
     5161 gcgcgggaga agcccctggg cctccggaga tgggggacac cccacgccag ttcggaggcg
     5221 cgaggccgcg ctcgggaggc gcgctccggg ggtgccgctc tcggggcggg ggcaaccggc
     5281 ggggtctttg tctgagccgg gctcttgcca atggggatcg cagggtgggc gcggcggagc
     5341 ccccgccagg cccggtgggg gctggggcgc cattgcgcgt gcgcgctggt cctttgggcg
     5401 ctaactgcgt gcgcgctggg aattggcgct aattgcgcgt gcgcgctggg actcaaggcg
     5461 ctaactgcgc gtgcgttctg gggcccgggg tgccgcggcc tgggctgggg cgaaggcggg
     5521 ctcggccgga aggggtgggg tcgccgcggc tcccgggcgc ttgcgcgcac ttcctgcccg
     5581 agccgctggc cgcccgaggg tgtggccgct gcgtgcgcgc gcgccgaccc ggcgctgttt
     5641 gaaccgggcg gaggcggggc tggcgcccgg ttgggagggg gttggggcct ggcttcctgc
     5701 cgcgcgccgc ggggacgcct ccgaccagtg tttgcctttt atggtaataa cgcggccggc
     5761 ccggcttcct ttgtccccaa tctgggcgcg cgccggcgcc ccctggcggc ctaaggactc
     5821 ggcgcgccgg aagtggccag ggcgggggcg acctcggctc acagcgcgcc cggctattct
     5881 cgcaactgac a
//
