LOCUS       sequence-354399-        7221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control of targeted optogenetic manipulation using TMP-Halo
            dimerization system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  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 7221)
  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-eDHFR
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..583
                     /label=chicken beta-actin promoter
     intron          584..1600
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1524..1546
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1608..1627
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1683..2390
                     /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(1828..1846)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2096..2115
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2409..2882
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             2883..2912
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3019..3038)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3084..3139
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3138..3157)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    3503..3536
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3549..3670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3586..3605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3640..3659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3689..3709)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3689..3707)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3728..3744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3728..3744)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3741..3763)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3752..3768
                     /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(3776..3782,3783..3800,3801..3806))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3821..3842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3959..3976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4130..4718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4210..4229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4889..5680,5681..5749))
                     /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     5512..5531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5750..5854)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5927..5975
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    6187..6220
                     /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             6225..6824
                     /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(6225..6244)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6721..6741
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6960..6977)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6966..7077
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggggggcgc gcgccrggsg gggsggggsg gggsgrgggg sggggsgggg sgaggcggag
      481 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      541 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
      601 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
      661 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg
      721 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg
      781 ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg
      841 gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg
      901 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
      961 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     1021 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
     1081 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
     1141 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
     1201 ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     1261 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     1321 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     1381 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     1441 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     1501 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     1561 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     1621 tattgtgctg tctcatcatt ttggcaaaga attgctcgac caccggcgcg ccaccggtca
     1681 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     1741 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     1801 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1861 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     1921 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     1981 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2041 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2101 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2161 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2221 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2281 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2341 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtataag gccggcctgg
     2401 agtctagaat cagtctgatt gcggcgttag cggtagatcg cgttatcggc atggaaaacg
     2461 ccatgccgtg gaacctgcct gccgatctcg cctggtttaa acgcaacacc ttaaataaac
     2521 ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca ggacgcaaaa
     2581 atattatcct cagcagtcaa ccgggtacgg acgatcgcgt aacgtgggtg aagtcggtgg
     2641 atgaagccat cgcggcgtgt ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg
     2701 tttatgaaca gttcttgcca aaagcgcaaa aactgtatct gacgcatatc gacgcagaag
     2761 tggaaggcga cacccatttc ccggattacg agccggatga ctgggaatcg gtattcagcg
     2821 aattccacga tgctgatgcg cagaactctc acagctattg ctttgagatt ctggagcggc
     2881 gggaacaaaa actcatctca gaagaggatc tttaattaag cgctcgaggc ggccgcaatt
     2941 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     3001 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     3061 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     3121 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     3181 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     3241 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     3301 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     3361 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     3421 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcaccg
     3481 tcgagctagc atatggatcc atataacttc gtataatgta tgctatacga agttattacg
     3541 ataccgtcaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3661 atgtatctta ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     3721 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3781 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3841 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3901 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3961 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4561 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4861 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4921 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4981 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5041 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5101 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5161 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5221 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5281 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5341 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5401 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5461 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5521 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5581 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5641 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5701 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5761 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5821 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaagct
     5881 taataaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5941 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     6001 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     6061 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     6121 ggcggcaggc cctgccatag ccactggccc cgtgggttag ggacggggtc ccccattcga
     6181 gaataaataa cttcgtataa agtatcctat acgaagttat caccatgacc gagtacaagc
     6241 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     6301 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg
     6361 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     6421 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     6481 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     6541 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     6601 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     6661 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     6721 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     6781 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     6841 cgcagcgccc gaccgaaagg agcgcacgac cccatggctc cgaccgaagc cacccggggc
     6901 ggccccgccg accccgcacc cgcccccgag gcccaccgac tctagagctc gctgatcagc
     6961 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     7021 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     7081 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     7141 ggattgggaa gacaatagca ctagttatta atagtaatca attacggggt cattagttca
     7201 tagcccatat atggagttcc g
//
