LOCUS       sequence-362451-        5750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the N-terminal tagging of beta-actin with mRuby2
            in human cells using CRISPR/Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5750)
  AUTHORS   Husser MC, Ozugergin I, Resta T, Martin VJJ, Piekny AJ
  TITLE     Cytokinetic diversity in mammalian cells is revealed by the 
            characterization of endogenous anillin, Ect2 and RhoA.
  JOURNAL   Open Biol. 2022 Nov;12(11):220247. doi: 10.1098/rsob.220247. Epub 
            2022 Nov 23.
  PUBMED    36416720
REFERENCE   2  (bases 1 to 5750)
  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"; journalName: "Open 
            Biol."; date: "2022-11"; volume: "12(11):220247. doi"; pages: " 
            10.1098/rsob.220247. Epub 2022 Nov 23"
COMMENT     Sequence Label: pODN-mR2-ACTB
FEATURES             Location/Qualifiers
     source          1..5750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     1528..1547
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1528..1546
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1608..1625
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1609..1625
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2642..3352
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     promoter        complement(join(4805..4811,4812..4829,4830..4835))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    4850..4871
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4991..5008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5162..5750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5242..5261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gccgcccctg cagccgaatt atattatttt tgccaaataa
     1261 tttttaacaa aagctctgaa gtcttcttca tttaaattct tagatgatac ttcatctgga
     1321 aaattgtccc aattagtagc atcacgctgt gagtaagttc taaaccattt ttttattgtt
     1381 gtattatctc taatcttact actcgatgag ttttcggtat tatctctatt tttaacttgg
     1441 agcaggttcc attcattgtt tttttcatca tagtgaataa aatcaactgc tttaacactt
     1501 gtgcctgaac accatatcca tccggcgtaa tacgactcac tatagggaga gcggccgcca
     1561 gatcttccgg atggctcgag tttttcagca agatccagtc acgacgttgt aaaacgacgg
     1621 ccagtgaatt gacgcgtatt ggatgctcga gcggccgcgg cggcgcccta taaaacccag
     1681 cggcgcgacg cgccaccacc gccgagaccg cgtccgcccc gcgagcacag agcctcgcct
     1741 ttgccgatcc gccgcccgtc cacacccgcc gccaggtaag cccggccagc cgaccggggc
     1801 aggcggctca cggcccggcc gcaggcggcc gcggcccctt cgcccgtgca gagccgccgt
     1861 ctgggccgca gcggggggcg catggggggg gaaccggacc gccgtggggg gcgcgggaga
     1921 agcccctggg cctccggaga tgggggacac cccacgccag ttcggaggcg cgaggccgcg
     1981 ctcgggaggc gcgctccggg ggtgccgctc tcggggcggg ggcaaccggc ggggtctttg
     2041 tctgagccgg gctcttgcca atggggatcg cagggtgggc gcggcggagc ccccgccagg
     2101 cccggtgggg gctggggcgc cattgcgcgt gcgcgctggt cctttgggcg ctaactgcgt
     2161 gcgcgctggg aattggcgct aattgcgcgt gcgcgctggg actcaaggcg ctaactgcgc
     2221 gtgcgttctg gggcccgggg tgccgcggcc tgggctgggg cgaaggcggg ctcggccgga
     2281 aggggtgggg tcgccgcggc tcccgggcgc ttgcgcgcac ttcctgcccg agccgctggc
     2341 cgcccgaggg tgtggccgct gcgtgcgcgc gcgccgaccc ggcgctgttt gaaccgggcg
     2401 gaggcggggc tggcgcccgg ttgggagggg gttggggcct ggcttcctgc cgcgcgccgc
     2461 ggggacgcct ccgaccagtg tttgcctttt atggtaataa cgcggccggc ccggcttcct
     2521 ttgtccccaa tctgggcgcg cgccggcgcc ccctggcggc ctaaggactc ggcgcgccgg
     2581 aagtggccag ggcgggggcg acctcggctc acagcgcgcc cggctattct cgcagctcac
     2641 catggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     2701 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     2761 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     2821 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     2881 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     2941 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     3001 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     3061 aaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     3121 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     3181 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     3241 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     3301 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca aggccggctc
     3361 cggtaccatg gatgacgaca ttgcagcgct cgtcgtcgac aacggctccg gcatgtgcaa
     3421 ggccggcttc gcgggcgacg atgccccccg ggccgtcttc ccctccatcg tggggcgccc
     3481 caggcaccag gtaggggagc tggctgggtg gggcagcccc gggagcgggc gggaggcaag
     3541 ggcgctttct ctgcacagga gcctcccggt ttccggggtg ggggctgcgc ccgtgctcag
     3601 ggcttcttgt cctttccttc ccagggcgtg atggtgggca tgggtcagaa ggattcctat
     3661 gtgggcgacg aggcccagag caagagaggc atcctcaccc tgaagtaccc catcgagcac
     3721 ggcatcgtca ccaactggga cgacatggag aaaatctggc accacacctt ctacaatgag
     3781 ctgcgtgtgg ctcccgagga gcaccccgtg ctgctgaccg aggcccccct gaaccccaag
     3841 gccaaccgcg agaagatgac ccaggtgagt ggcccgctac ctcttctggt ggccgcctcc
     3901 ctccttcctg gcctcccgga gctgcgccct ttctcactgg ttctctcttc tgccgttttc
     3961 cgtaggactc tcttctctga cctgagtctc ctttggaact ctgcaggttc tatttgcttt
     4021 ttcccagatg agctcttttt ctggtgtttg tctctctgac taggtgtcta agacagtgtt
     4081 gtgggtgtag gtactaacac tggctcgtgt gacaaggcca tgaggctggt gtaaagcggc
     4141 cttggagtgt gtattaagta ggcgcacagt aggtctgaac agactcccca tcccaagacc
     4201 ccagcacact tagccgtgtt ctttgcactt tctgcatgtc ccccgtctgg cctggctgtc
     4261 cccagtggct tccccagtgt gacatggtgt atctctgcct tacagatcat gtttgagacc
     4321 ttcaacaccc cagccatgta cgttgctatc caggctgtgc tatccctgta atctttctag
     4381 aagatctcct acaatattct cagctgccat ggaaaatcga tgttcttctt ttattctctc
     4441 aagattttca ggctgtatat taaaacttat attaagaact atgctaacca cctcatcagg
     4501 aaccgttgta ggtggcgtgg gttttcttgg caatcgactc tcatgaaaac tacgagctaa
     4561 atattcaata tgttcctctt gaccaacttt attctgcatt ttttttgaac gaggtttaga
     4621 gcaagcttca ggaaactgag acaggaattt tattaaaaat ttaaattttg aagaaagttc
     4681 agggttaata gcatccattt tttgctttgc aagttcctca gcattcttaa caaaagacgt
     4741 ctcttttgac atgtttaaag tttaaacctc ctgtgtgaaa ttattatccg ctcataattc
     4801 cacacattat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4861 aactcacatt aattgcgttg cgctcactgc caattgcttt ccagtcggga aacctgtcgt
     4921 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4981 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5041 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5101 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5161 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5281 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5341 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5401 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5461 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5521 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5581 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5641 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5701 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
//
