LOCUS       sequence-338566-        7686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SPELL Vav2(DH-PH-ZnF). Two components cloned into the same plasmid 
            with T2A/P2A sequence. Component 2-T2A-P2A-Component 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7686)
  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 7686)
  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-pBabe-Vav2(DPZ) (Component 2-T2AP2A-Comp...
FEATURES             Location/Qualifiers
     source          1..7686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..234
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     185..205
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     437..459
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     521..540
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     582..601
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        582..600
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    601..625
                     /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        641..750
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             768..797
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             828..1103
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRIS"
     CDS             1821..1874
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1884..1940
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1947..1970
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1992..2001
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1998..2705
                     /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(2143..2161)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2411..2430
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3519..3542
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3632..3651)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3697..3752
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3751..3770)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3840..3887
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3900..4605
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3988..4360
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4017..4605)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4614..4647)
                     /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(4815..5402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4894..4913)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5415..6206,6207..6275))
                     /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     6038..6057
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     6518..7345
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             6518..6642
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6680..7285)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        join(7413..7686,1..30)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta caccaatggg
       61 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      121 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg
      181 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag acgtcgttta
      241 gtgaaccgtc agatcactag atgctttatt gcggtagttt atcacagtta aattgctaac
      301 gccagtctcg aacttaacgt gcagaagttg gtcgtgaggc actgggcagg taagtatcgg
      361 gccctttgtg cggggggagc ggctcggggc tgtccgcggg gggacggctg ccttcggggg
      421 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
      481 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
      541 ctgtctcatc attttggcaa agaattggat cggaccgaaa ttaatacgac tcactatagg
      601 ggaattgtga gcggataaca attccccgga gttaatccgg gacctttaat tcaacccaac
      661 acaatatatt atagttaaat aagaattatt atcaaatcat ttgtatatta attaaaatac
      721 tatactgtaa attacatttt atttacaatc aaaggagata taccatggag cagaagctga
      781 tctccgagga ggacctggga tctggatccg aattcatggc ttctagaatc ctctggcatg
      841 agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga
      901 aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacggggc ccccagactc
      961 tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt
     1021 gcaggaagta catgaaatca gggaatgtca aggacctcct ccaagcctgg gacctctatt
     1081 atcatgtgtt ccgacgaatc tcaggtggat caggtggcgc ggccgcgcga ccagaaagac
     1141 aacagctcaa agaagccctg gaagccatgc aggacttggc catgtacatc aatgaagtga
     1201 agcgggacaa ggagaccttg aagaagatta gcgagttcca gtgctccata gaaaacctgc
     1261 aagtgaagct ggaggaattt gggaggccaa agattgacgg ggagcttaaa gtccggtcca
     1321 tagtcaacca caccaagcaa gacaggtacc tgttcctatt tgacaaggtg gtcatcgtgt
     1381 gcaagaggaa gggctacagc tatgagctga aggaggtcat tgagctgctc ttccacaaga
     1441 tgaccgatga cccgatgcac aacaaggaca tcaagaagtg gtcctatggc ttctacctga
     1501 ttcacctcca aggaaagcaa ggctttcagt tcttctgcaa gacggaagac atgaagcgga
     1561 agtggatgga gcagttcgag atggccatgt caaacatcaa gccagataag gccaatgcca
     1621 accatcatag cttccagatg tacacattcg acaagactac caactgcaaa gcctgcaaga
     1681 tgtttctcag gggtaccttc taccagggat acctgtgtac cagatgtggc gtcggggcac
     1741 acaaggaatg cctggaggtg atccccccct gcaagatgag ttcacctgca gatgtggatg
     1801 ctcctggagc aggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg
     1861 agaatcctgg cccaggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     1921 tggaggagaa ccctggacct actagtgact ataaggacga tgatgacaaa ggatctggat
     1981 ccgaattcgc cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     2041 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     2101 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     2161 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     2221 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     2281 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     2341 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     2401 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     2461 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     2521 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     2581 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     2641 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     2701 acaagtccgg aatcaaggtg gaggtgcagc agcccatgaa aatgggcatg actgaggacg
     2761 acaagagaag ctgctgcttg ttagagattc aggagaccga ggccaagtac taccgcaccc
     2821 tggaggacat tgagaagaac tacatgggtc ccttgcggct ggtgctgagc ccggcggata
     2881 tggctgctgt cttcatcaac ctggaggacc tcatcaaggt gcatcacagc tttctgcgag
     2941 ccatcgatgt gtccatgatg gctggtggca gtaccctggc taaggtcttt ctggagttta
     3001 aggaaaggct cctgatctat ggagagtact gtagccacat ggaacacgct cagagtacac
     3061 tgaaccagct cctcgccagc cgagaggact tcaggcagaa agtggaggag tgcacactca
     3121 gggttcagga tggcaagttc aagctgcaag acctgctggt ggtgcccatg caacgggtgc
     3181 tgaagtacca cctgctgctc aaggagctcc tgagccattc tgcaggtgga tcaggtggcg
     3241 cggccgcgac ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaatttgatt
     3301 cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag
     3361 gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc
     3421 cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg
     3481 tcttcgatgt ggagcttcta aaactggaat gactcgagca ccaccatcac catcaccatc
     3541 actaagtgat taacctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     3601 tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg
     3661 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3721 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     3781 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac
     3841 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgcg
     3901 gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa aggagtttgc accagacgca
     3961 cctctgttca ctggtccggc gtattaaaac acgatacatt gttattagta catttattaa
     4021 gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca
     4081 ttaaatttat aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct
     4141 ttatatctga atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt
     4201 tcaaacaagg gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac
     4261 gccacaaaac ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt
     4321 ttgttttgta ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca
     4381 tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt taaatagagc ttggacatat
     4441 ttaacatcgg gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg
     4501 ttagaagttg cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg
     4561 gctaacacgt ccgcgatcaa atttgtagtt gagctttttg gaattgcgat cgcataactt
     4621 cgtatagcat acattatacg aagttataag ctcggaacgc tgcgctcggt cgttcggctg
     4681 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4741 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4801 gcgttgctgg cgtttttcca taggctccgc cccctgacga gcatcacaaa aatcgacgct
     4861 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4921 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4981 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5041 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5101 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5161 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5221 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5281 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5341 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5401 aagaagatcc tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5461 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5521 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5581 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5641 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5701 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5761 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5821 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5881 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5941 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6001 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6061 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6121 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6181 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6241 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6301 agcatttatc agggttattg tctcatgtcc gcgcgtttcc tgcatctttt aatcaaatcc
     6361 caagatgtgt ataaacgcgc cggtatgtac aggaagaggt ttatactaaa ctgttacatt
     6421 gcaaacgtgg tttcgtgtgc caagtgtgaa aaccgatgtt taatcaaggc tctgacgcat
     6481 ttctacaacc acgactccaa gtgtgtgggt gaagtcatgc atcttttaat caaatcccaa
     6541 gatgtgtata aaccaccaaa ctgccaaaaa atgaaaactg tcgacaagct ctgtccgttt
     6601 gctggcaact gcaagggtct caatcctatt tgtaattatt gaataataaa acaattataa
     6661 atgtcaaatt tgttttttat taacgataca aaccaaacgc aacaagaaca tttgtagtat
     6721 tatctataat tgaaaacgcg tagttataat cgctgaggta atatttaaaa tcattttcaa
     6781 atgattcaca gttaatttgc gacaatataa ttttattttc acataaacta gacgccttgt
     6841 cgtcttcttc ttcgtattcc ttctcttttt catttttctc ttcataaaaa ttaacatagt
     6901 tattatcgta tccatatatg tatctatcgt atagagtaaa ttttttgttg tcataaatat
     6961 atatgtcttt tttaatgggg tgtatagtac cgctgcgcat agtttttctg taatttacaa
     7021 cagtgctatt ttctggtagt tcttcggagt gtgttgcttt aattattaaa tttatataat
     7081 caatgaattt gggatcgtcg gttttgtaca atatgttgcc ggcatagtac gcagcttctt
     7141 ctagttcaat tacaccattt tttagcagca ccggattaac ataactttcc aaaatgttgt
     7201 acgaaccgtt aaacaaaaac agttcacctc ccttttctat actattgtct gcgagcagtt
     7261 gtttgttgtt aaaaataaca gccattgtaa tgagacgcac aaactaatat cacaaactgg
     7321 aaatgtctat caatatatag ttgctgatgg ccggcctatt aatagtaatc aattacgggg
     7381 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     7441 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     7501 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     7561 cacttggcag tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac
     7621 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg
     7681 cagtac
//
