LOCUS       sequence-223594-        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     81..103
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     95..112
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     96..112
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     119..138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        119..137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     170..186
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     170..186
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1171..1902
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     promoter        complement(3199..3217)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3238..3254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3238..3254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3251..3273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3262..3278
                     /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(3286..3292,3293..3310,3311..3316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3331..3352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3469..3486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3640..4228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3720..3739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4399..5190,5191..5259))
                     /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     5022..5041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5260..5364)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5390..5845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5477..5496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5687..5708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
       61 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta
      121 atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct ctagaactag
      181 tggatccggc ggcgccctat aaaacccagc ggcgcgacgc gccaccaccg ccgagaccgc
      241 gtccgccccg cgagcacaga gcctcgcctt tgccgatccg ccgcccgtcc acacccgccg
      301 ccaggtaagc ccggccagcc gaccggggca ggcggctcac ggcccggccg caggcggccg
      361 cggccccttc gcccgtgcag agccgccgtc tgggccgcag cggggggcgc atgggggggg
      421 aaccggaccg ccgtgggggg cgcgggagaa gcccctgggc ctccggagat gggggacacc
      481 ccacgccagt tcggaggcgc gaggccgcgc tcgggaggcg cgctccgggg gtgccgctct
      541 cggggcgggg gcaaccggcg gggtctttgt ctgagccggg ctcttgccaa tggggatcgc
      601 agggtgggcg cggcggagcc cccgccaggc ccggtggggg ctggggcgcc attgcgcgtg
      661 cgcgctggtc ctttgggcgc taactgcgtg cgcgctggga attggcgcta attgcgcgtg
      721 cgcgctggga ctcaaggcgc taactgcgcg tgcgttctgg ggcccggggt gccgcggcct
      781 gggctggggc gaaggcgggc tcggccggaa ggggtggggt cgccgcggct cccgggcgct
      841 tgcgcgcact tcctgcccga gccgctggcc gcccgagggt gtggccgctg cgtgcgcgcg
      901 cgccgacccg gcgctgtttg aaccgggcgg aggcggggct ggcgcccggt tgggaggggg
      961 ttggggcctg gcttcctgcc gcgcgccgcg gggacgcctc cgaccagtgt ttgcctttta
     1021 tggtaataac gcggccggcc cggcttcctt tgtccccaat ctgggcgcgc gccggcgccc
     1081 cctggcggcc taaggactcg gcgcgccgga agtggccagg gcgggggcga cctcggctca
     1141 cagcgcgccc ggctattctc gcaactgaca atggtgtcta agggcgaaga gctgattaag
     1201 gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc
     1261 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     1321 gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc
     1381 agaaccttca tcaaccacac ccagggcatc cccgatttct ttaagcagtc cttccctgag
     1441 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     1501 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     1561 ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagatg
     1621 ctgtaccccg ctgacggcgg cctggaaggc agaaccgaca tggccctgaa gctcgtgggc
     1681 gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac
     1741 ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac
     1801 aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc
     1861 aaactggggc acaaacttaa tggcatggac gagctgtaca agtctagagc cggctccggt
     1921 accgatgatg atatcgcagc gctcgtcgtc gacaacggct ccggcatgtg caaggccggc
     1981 ttcgcgggcg acgatgcccc ccgggccgtc ttcccctcca tcgtggggcg ccccaggcac
     2041 caggtagggg agctggctgg gtggggcagc cccgggagcg ggcgggaggc aagggcgctt
     2101 tctctgcaca ggagcctccc ggtttccggg gtgggggctg cgcccgtgct cagggcttct
     2161 tgtcctttcc ttcccagggc gtgatggtgg gcatgggtca gaaggattcc tatgtgggcg
     2221 acgaggccca gagcaagaga ggcatcctca ccctgaagta ccccatcgag cacggcatcg
     2281 tcaccaactg ggacgacatg gagaaaatct ggcaccacac cttctacaat gagctgcgtg
     2341 tggctcccga ggagcacccc gtgctgctga ccgaggcccc cctgaacccc aaggccaacc
     2401 gcgagaagat gacccaggtg agtggcccgc tacctcttct ggtggccgcc tccctccttc
     2461 ctggcctccc ggagctgcgc cctttctcac tggttctctc ttctgccgtt ttccgtagga
     2521 ctctcttctc tgacctgagt ctcctttgga actctgcagg ttctatttgc tttttcccag
     2581 atgagctctt tttctggtgt ttgtctctct gactaggtgt ctaagacagt gttgtgggtg
     2641 taggtactaa cactggctcg tgtgacaagg ccatgaggct ggtgtaaagc ggccttggag
     2701 tgtgtattaa gtaggcgcac agtaggtctg aacagactcc ccatcccaag accccagcac
     2761 acttagccgt gttctttgca ctttctgcat gtcccccgtc tggcctggct gtccccagtg
     2821 gcttccccag tgtgacatgg tgtatctctg ccttacagat catgtttgag accttcaaca
     2881 ccccagccat gtacgttgct atccaggctg tgctatccct ttgaggaaga tgaactgaga
     2941 tgtgtcaggg tgacttattt ccatcatcgt ccttagggga gcttgggtag gggcaaggcg
     3001 tgtagctggg acctaggtcc agacccctgg ctctgccact gaacggctca ggtgctttgg
     3061 gcagttactc cccggcctca ctttgcacgt gtgcttacct agtggagaca aaagtacata
     3121 cctcggtaga gcgcgcacgc ctgtaacccc aggttaactc tctaacctcg agggggggcc
     3181 cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt aatcatggtc
     3241 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3301 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3361 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4081 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4321 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5341 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta cgcgccctgt
     5401 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5461 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5521 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5581 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5641 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5701 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5761 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5821 aacaaaatat taacgcttac aatttcccat tcgccattca ggctgcgcaa ctgttgggaa
     5881 gggcgatcgg t
//
