LOCUS       sequence-338474-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SPELL ITSN1(DH-PH) component 1. mCherry-ITSN(N-lobe) fused to iFKBP.
            Split at loop 4 of DH domain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  AUTHORS   Dagliyan O, Krokhotin A, Ozkan-Dagliyan I, Deiters A, Der CJ, Hahn 
            KM, Dokholyan NV
  TITLE     Computational design of chemogenetic and optogenetic split proteins.
  JOURNAL   Nat Commun. 2018 Oct 2;9(1):4042. doi: 10.1038/s41467-018-06531-4.
  PUBMED    30279442
REFERENCE   2  (bases 1 to 6516)
  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: "Nat 
            Commun."; date: "2018-10-2"; volume: "9(1):4042. doi"; pages: " 
            10.1038/s41467-018-06531-4"
COMMENT     Sequence Label: SPELL-ITSN1(DH-PH) Component 1
FEATURES             Location/Qualifiers
     source          1..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..240
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     191..211
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     443..465
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     527..546
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     588..607
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        588..606
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    607..631
                     /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        647..756
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             774..797
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      819..828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             825..1532
                     /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(970..988)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1238..1257
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2355..2378
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2468..2487)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2533..2588
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2587..2606)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2676..2723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2736..3441
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2824..3196
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2853..3441)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3450..3483)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(3651..4238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3730..3749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4251..5042,5043..5111))
                     /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     4874..4893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     5354..6181
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5354..5478
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5516..6121)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        join(6249..6516,1..36)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg gcagtacacc
       61 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      121 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc
      181 gccccgttga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagacgt
      241 cgtttagtga accgtcagat cactagatgc tttattgcgg tagtttatca cagttaaatt
      301 gctaacgcca gtctcgaact taacgtgcag aagttggtcg tgaggcactg ggcaggtaag
      361 tatcgggccc tttgtgcggg gggagcggct cggggctgtc cgcgggggga cggctgcctt
      421 cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc
      481 tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt
      541 attgtgctgt ctcatcattt tggcaaagaa ttggatcgga ccgaaattaa tacgactcac
      601 tataggggaa ttgtgagcgg ataacaattc cccggagtta atccgggacc tttaattcaa
      661 cccaacacaa tatattatag ttaaataaga attattatca aatcatttgt atattaatta
      721 aaatactata ctgtaaatta cattttattt acaatcaaag gagatatacc atggactata
      781 aggacgatga tgacaaagga tctggatccg aattcgccgc caccatggtg agcaagggcg
      841 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      901 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
      961 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1021 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1081 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1141 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1201 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1261 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1321 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1381 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1441 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1501 actccaccgg cggcatggac gagctgtaca agtccggaat ggatatgttg accccaactg
     1561 aaagaaagcg acaaggatac atccacgagc tcattgtcac cgaggagaac tatgtgaatg
     1621 acctgcagct ggtcacagag atttttcaaa aacccctgat ggagtctgag ctgctgacag
     1681 aaaaagaggt tgctatgatt tttgtgaact ggaaggagct gattatgtgt aatatcaaac
     1741 tactaaaagc gctgagagtc cgcaagaaga tgtccgggga gaagatgcct gtgaagatga
     1801 ttggagacat cctgagcgca cagctgccgc acatgcagcc ctacatccgc ttctgcagcc
     1861 gccagctcaa cggggctgcc ctgatccagc agaagacgga cgaggcccca gacttcaagg
     1921 agttcgtcaa aagattggca atggatcctc ggtgtaaagg gatgccactc tctagtttta
     1981 tactgaagcc tatgcaacgg gtaacaagat acccactgat cattaaaaat atcctggaaa
     2041 acacccctga acattctgca ggtggatcag gtggcgcggc cgcgacctgc gtggtgcact
     2101 acaccgggat gcttgaagat ggaaagaaat ttgattcctc ccgggacaga aacaagccct
     2161 ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga
     2221 tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg
     2281 ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac
     2341 tggaatgact cgagcaccac catcaccatc accatcacta agtgattaac ctcaggtgca
     2401 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     2461 agatcgatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
     2521 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
     2581 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
     2641 tggtttagag tttggcaaca tatgcccata tgtaactagc ataacccctt ggggcctcta
     2701 aacgggtctt gaggggtttt ttgctgaaag catgcggagg aaattctcct tgaagtttcc
     2761 ctggtgttca aagtaaagga gtttgcacca gacgcacctc tgttcactgg tccggcgtat
     2821 taaaacacga tacattgtta ttagtacatt tattaagcgc tagattctgt gcgttgttga
     2881 tttacagaca attgttgtac gtattttaat aattcattaa atttataatc tttagggtgg
     2941 tatgttagag cgaaaatcaa atgattttca gcgtctttat atctgaattt aaatattaaa
     3001 tcctcaatag atttgtaaaa taggtttcga ttagtttcaa acaagggttg tttttccgaa
     3061 ccgatggctg gactatctaa tggattttcg ctcaacgcca caaaacttgc caaatcttgt
     3121 agcagcaatc tagctttgtc gatattcgtt tgtgttttgt tttgtaataa aggttcgacg
     3181 tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac tgtcgttagt gtacaattga
     3241 ctcgacgtaa acacgttaaa tagagcttgg acatatttaa catcgggcgt gttagcttta
     3301 ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat
     3361 tttgccatag ccacacgacg cctattaatt gtgtcggcta acacgtccgc gatcaaattt
     3421 gtagttgagc tttttggaat tgcgatcgca taacttcgta tagcatacat tatacgaagt
     3481 tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3541 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3601 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3661 ctccgccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3721 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3781 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3841 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3901 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3961 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4021 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4081 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4141 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4201 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg ttaccaatgc
     4261 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4321 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4381 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4441 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4501 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4561 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4621 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4681 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4741 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4801 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4861 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4921 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4981 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5041 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5101 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5161 atgtccgcgc gtttcctgca tcttttaatc aaatcccaag atgtgtataa acgcgccggt
     5221 atgtacagga agaggtttat actaaactgt tacattgcaa acgtggtttc gtgtgccaag
     5281 tgtgaaaacc gatgtttaat caaggctctg acgcatttct acaaccacga ctccaagtgt
     5341 gtgggtgaag tcatgcatct tttaatcaaa tcccaagatg tgtataaacc accaaactgc
     5401 caaaaaatga aaactgtcga caagctctgt ccgtttgctg gcaactgcaa gggtctcaat
     5461 cctatttgta attattgaat aataaaacaa ttataaatgt caaatttgtt ttttattaac
     5521 gatacaaacc aaacgcaaca agaacatttg tagtattatc tataattgaa aacgcgtagt
     5581 tataatcgct gaggtaatat ttaaaatcat tttcaaatga ttcacagtta atttgcgaca
     5641 atataatttt attttcacat aaactagacg ccttgtcgtc ttcttcttcg tattccttct
     5701 ctttttcatt tttctcttca taaaaattaa catagttatt atcgtatcca tatatgtatc
     5761 tatcgtatag agtaaatttt ttgttgtcat aaatatatat gtctttttta atggggtgta
     5821 tagtaccgct gcgcatagtt tttctgtaat ttacaacagt gctattttct ggtagttctt
     5881 cggagtgtgt tgctttaatt attaaattta tataatcaat gaatttggga tcgtcggttt
     5941 tgtacaatat gttgccggca tagtacgcag cttcttctag ttcaattaca ccatttttta
     6001 gcagcaccgg attaacataa ctttccaaaa tgttgtacga accgttaaac aaaaacagtt
     6061 cacctccctt ttctatacta ttgtctgcga gcagttgttt gttgttaaaa ataacagcca
     6121 ttgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc
     6181 tgatggccgg cctattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     6241 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     6301 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     6361 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     6421 catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     6481 gcccagtaca tgaccttacg ggactttcct acttgg
//
