LOCUS       sequence-348684-        7536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  INBox TSS(892-894)-mutant replaced by AAA responsible for targeted 
            Aurora B recruitment without activation using TMP-Halo dimerization 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7536)
  AUTHORS   Chen GY, Renda F, Zhang H, Gokden A, Wu DZ, Chenoweth DM, Khodjakov 
            A, Lampson MA
  TITLE     Tension promotes kinetochore-microtubule release by Aurora B kinase.
  JOURNAL   J Cell Biol. 2021 Jun 7;220(6). pii: 212027. doi: 
            10.1083/jcb.202007030.
  PUBMED    33904910
REFERENCE   2  (bases 1 to 7536)
  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: "J Cell 
            Biol."; date: "2021-06-7"; volume: "220(6). pii: 212027. doi"; 
            pages: " 10.1083/jcb.202007030"
COMMENT     Sequence Label: CAG.mCherry-INBox(AAA)-eDHFR
FEATURES             Location/Qualifiers
     source          1..7536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    52..100
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    312..345
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             350..949
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(350..369)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     846..866
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1085..1102)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1091..1202
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1347..1650
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1652..1929
                     /label=chicken beta-actin promoter
     intron          1930..2946
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2870..2892
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2954..2973
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3029..3736
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3174..3192)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3442..3461
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4070..4543
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             4544..4573
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(4680..4699)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4745..4800
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4799..4818)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    5164..5197
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5210..5331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5247..5266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5301..5320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5350..5370)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5350..5368)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5389..5405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5389..5405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5402..5424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5413..5429
                     /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(5437..5443,5444..5461,5462..5467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5482..5503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5620..5637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5791..6379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5871..5890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6550..7341,7342..7410))
                     /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     7173..7192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7411..7515)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagcttaata aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
       61 aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
      121 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
      181 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
      241 gtcggggcgg caggccctgc catagccact ggccccgtgg gttagggacg gggtccccca
      301 ttcgagaata aataacttcg tataaagtat cctatacgaa gttatcacca tgaccgagta
      361 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc
      421 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga
      481 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
      541 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
      601 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
      661 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
      721 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
      781 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
      841 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
      901 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca
      961 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat ggctccgacc gaagccaccc
     1021 ggggcggccc cgccgacccc gcacccgccc ccgaggccca ccgactctag agctcgctga
     1081 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1141 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1201 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1261 ggggaggatt gggaagacaa tagcactagt tattaatagt aatcaattac ggggtcatta
     1321 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     1381 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     1441 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     1501 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1561 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1621 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
     1681 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
     1741 tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc crggsggggs ggggsggggs
     1801 grggggsggg gsggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
     1861 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
     1921 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
     1981 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
     2041 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
     2101 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
     2161 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
     2221 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     2281 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     2341 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     2401 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     2461 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     2521 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     2581 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     2641 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     2701 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     2761 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     2821 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     2881 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     2941 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg
     3001 ctcgaccacc ggcgcgccac cggtcaccat ggtgagcaag ggcgaggagg ataacatggc
     3061 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     3121 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     3181 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     3241 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     3301 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     3361 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     3421 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     3481 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     3541 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     3601 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     3661 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     3721 ggacgagctg tataagggta ccccagataa ctacgggatg gatctgaata gcgacgactc
     3781 caccgatgat gaggcccatc cccggaagcc catccccacc tgggcccgag gcaccccgct
     3841 cagccaggct atcattcacc agtactacca cccaccgaac cttctggagc tctttggaac
     3901 cattctccca ctggacttgg aggatatctt caagaagagc aagccccgct atcacaagcg
     3961 cgccgccgct gctgtctgga actcaccgcc cctgcagggc gccagggtcc ccagcagcct
     4021 ggcctacagc ctgaagaagc acggatccac cggatccctg gagtctagaa tcagtctgat
     4081 tgcggcgtta gcggtagatc gcgttatcgg catggaaaac gccatgccgt ggaacctgcc
     4141 tgccgatctc gcctggttta aacgcaacac cttaaataaa cccgtgatta tgggccgcca
     4201 tacctgggaa tcaatcggtc gtccgttgcc aggacgcaaa aatattatcc tcagcagtca
     4261 accgggtacg gacgatcgcg taacgtgggt gaagtcggtg gatgaagcca tcgcggcgtg
     4321 tggtgacgta ccagaaatca tggtgattgg cggcggtcgc gtttatgaac agttcttgcc
     4381 aaaagcgcaa aaactgtatc tgacgcatat cgacgcagaa gtggaaggcg acacccattt
     4441 cccggattac gagccggatg actgggaatc ggtattcagc gaattccacg atgctgatgc
     4501 gcagaactct cacagctatt gctttgagat tctggagcgg cgggaacaaa aactcatctc
     4561 agaagaggat ctttaattaa gcgctcgagg cggccgcaat tcactcctca ggtgcaggct
     4621 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     4681 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     4741 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     4801 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     4861 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     4921 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     4981 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     5041 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     5101 gctgtccctc ttctcttatg gagatccctc gacctgcacc gtcgagctag catatggatc
     5161 catataactt cgtataatgt atgctatacg aagttattac gataccgtca acttgtttat
     5221 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5281 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aggtacccag
     5341 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     5401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5581 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5641 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5701 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5761 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5821 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5881 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5941 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6001 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6061 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7501 aataggggtt ccgcgcacat ttccccgaaa agtgcc
//
