LOCUS       sequence-373475-        6729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the stable expression of a mRuby2-CAAX fusion in
            human cells using CRISPR/Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6729)
  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 6729)
  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-AAVS1-mR2-CAAX
FEATURES             Location/Qualifiers
     source          1..6729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    880..901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(916..921,922..939,940..946)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    1575..2378
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     enhancer        2430..2733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2734..2937
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2888..2908
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    2960..3040
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      3042..3051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3048..3758
                     /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"
     polyA_signal    3980..4101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4071..4090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    4152..4988
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5183..5202)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5184..5202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5508..5526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5594..5698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5699..5767,5768..6559)
                     /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     complement(5917..5936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcaattg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      901 ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg gaattatgag
      961 cggataataa tttcacacag gaggtttaaa ctttaaacat gtcaaaagag acgtcttttg
     1021 ttaagaatgc tgaggaactt gcaaagcaaa aaatggatgc tattaaccct gaactttctt
     1081 caaaatttaa atttttaata aaattcctgt ctcagtttcc tgaagcttgc tctaaacctc
     1141 gttcaaaaaa aatgcagaat aaagttggtc aagaggaaca tattgaatat ttagctcgta
     1201 gttttcatga gagtcgattg ccaagaaaac ccacgccacc tacaacggtt cctgatgagg
     1261 tggttagcat agttcttaat ataagtttta atatacagcc tgaaaatctt gagagaataa
     1321 aagaagaaca tcgattttcc atggcagctg agaatattgt aggagatctt ctagaaagat
     1381 ctcagtctgg tctatctgcc tggccctggc cattgtcact ttgcgctgcc ctcctctcgc
     1441 cccgagtgcc cttgctgtgc cgccggaact ctgccctcta acgctgccgt ctctctcctg
     1501 agtccggacc actttgagct ctactggctt ctgcgccgcc tctggcccac tgcttcccct
     1561 tcccaggcag gtcctgcttt ctctgacctg cattctctcc cctgggcctg tgccgctttc
     1621 tgtctgcagc ttgtggtctg ggtcacctct acggctggcc cagatccttc cctgccgcat
     1681 ccttcaggtt ccgtcttcct ccactccctc ttccccttgc tctctgctgt gttgctgccc
     1741 aaggatgctc tttccggagc acttccttct cggcgctgca ccacgtgatg tcctctgagc
     1801 ggatcctccc cgtgtctggg tcctctccgg gcatctctcc tccctcaccc aaccccatgc
     1861 cgtcttcact cgctgggttc ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt
     1921 tcttaggatg gccttctccg acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg
     1981 cctgcatcat caccgttttt ctggacaacc ccaaagtacc ccgtctccct ggctttagcc
     2041 acctctccat cctcttgctt tctttgcctg gacaccccgt tctcctgtgg attcgggtca
     2101 cctctcaccc ctttcatttg ggcagctccc ctacccccct tacctctcta gtctgtgcta
     2161 gctcttccag ccccctgtca tggcatcttc caggggtccg agagctcagc tagtcttctt
     2221 cctccaaccc gggcccctat gtccacttca ggacagcatg tttgctgcct ccagggatcc
     2281 tgtgtccccg agctgggacc accttatatt cccagggccg gttaatgtgg ctctggttct
     2341 gggtactttt atctgtcccc tcgaccacac agtggggcct agtaatcaat tacggggtca
     2401 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     2461 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     2521 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     2581 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2641 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2701 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2761 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2821 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2881 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     2941 ttagtgaacc gtcagatccg ctagcgctac cggactcaga tctcgagctc aagcttcgaa
     3001 ttctgcagtc gacggtaccg cgggcccggg atccaccggt cgccaccatg gtgtctaagg
     3061 gcgaagagct gatcaaggaa aatatgcgta tgaaggtggt catggaaggt tcggtcaacg
     3121 gccaccaatt caaatgcaca ggtgaaggag aaggcaatcc gtacatggga actcaaacca
     3181 tgaggatcaa agtcatcgag ggaggacccc tgccatttgc ctttgacatt cttgccacgt
     3241 cgttcatgta tggcagccgt acttttatca agtacccgaa aggcattcct gatttcttta
     3301 aacagtcctt tcctgagggt tttacttggg aaagagttac gagatacgaa gatggtggag
     3361 tcgtcaccgt catgcaggac accagccttg aggatggctg tctcgtttac cacgtccaag
     3421 tcagaggggt aaactttccc tccaatggtc ccgtgatgca gaagaaaacc aagggttggg
     3481 agcctaatac agagatgatg tatccagcag atggtggtct gaggggatac actcatatgg
     3541 cactgaaagt tgatggtggt ggccatctgt cttgctcttt cgtaacaact tacaggtcaa
     3601 aaaagaccgt cgggaacatc aagatgcccg gtatccatgc cgttgatcac cgcctggaaa
     3661 ggttagagga aagtgacaat gaaatgttcg tagtacaacg cgaacacgca gttgccaagt
     3721 tcgccgggct tggtggtggg atggacgagc tgtacaagtc cggactcaga tctcgagctc
     3781 aagcttcgaa ttcaaagatg agcaaagatg gtaaaaagaa gaaaaagaag tcaaagacaa
     3841 agtgtgtaat tatgtaaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     3901 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3961 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4021 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4081 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     4141 cgttaaattt tactagggac aggcttagtg acagaaaagc cccatcctta ggcctcctcc
     4201 ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat tccttatctg
     4261 gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct aaggtttgct
     4321 tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg gagggaaggg
     4381 ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct cccagaacct
     4441 gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc agcttcctta
     4501 cacttcccaa gaggagaagc agtttggaaa aacaaaatca gaataagttg gtcctgagtt
     4561 ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg tgcgtcagtt
     4621 ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg tgaggagggg
     4681 ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt caccaggtcg
     4741 tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag tccccagtgc
     4801 tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc cctgctctgg
     4861 gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct
     4921 tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg ggttcctggc
     4981 tctgctcttc agactgagcc ccgttcccct gcatccccgt tcccctgcat cccccagagg
     5041 ccccaggcca cctacttggc ctggacccca cgagaggcca ccccagccct gtctaccagg
     5101 ctgccttttg ggtggattct cctccaactg tggggatctt gctgaaaaac tcgagccatc
     5161 cggaagatct ggcggccgct ctccctatag tgagtcgtat tacgccggat ggatatggtg
     5221 ttcaggcaca agtgttaaag cagttgattt tattcactat gatgaaaaaa acaatgaatg
     5281 gaacctgctc caagttaaaa atagagataa taccgaaaac tcatcgagta gtaagattag
     5341 agataataca acaataaaaa aatggtttag aacttactca cagcgtgatg ctactaattg
     5401 ggacaatttt ccagatgaag tatcatctaa gaatttaaat gaagaagact tcagagcttt
     5461 tgttaaaaat tatttggcaa aaataatata attcggctgc aggggcggcc tcgtgatacg
     5521 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5581 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5641 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5701 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5761 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5821 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5881 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5941 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6001 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6061 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6121 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6181 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6241 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6301 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6361 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6421 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6481 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6541 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6601 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6661 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6721 aggatcttc
//
