LOCUS       sequence-382141-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For use in light-induced Rab8a protein inactivation through the 
            interaction with CRY2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  AUTHORS   Aljiboury AA, Ingram E, Krishnan N, Ononiwu F, Pal D, Manikas J, 
            Taveras C, Hall NA, Da Silva J, Freshour J, Hehnly H
  TITLE     Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during 
            zebrafish left-right organizer development.
  JOURNAL   PLoS Genet. 2023 May 15;19(5):e1010765. doi: 
            10.1371/journal.pgen.1010765. eCollection 2023 May.
  PUBMED    37186603
REFERENCE   2  (bases 1 to 5960)
  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: "PLoS 
            Genet."; date: "2023-05-15"; volume: "19(5):e1010765. doi"; pages: "
            10.1371/journal.pgen.1010765. eCollection 2023 May"
COMMENT     Sequence Label: pCS2-CIB1-mCherry-Rab8A
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             89..598
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCN
                     EAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     regulatory      611..620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             617..1324
                     /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(762..780)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1030..1049
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2012..2146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2036..2055)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2090..2109
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2259..2279)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2259..2277)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2298..2314)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2298..2314)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2311..2333)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2322..2338
                     /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(2346..2352,2353..2370,2371..2376))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2391..2412
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2529..2546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2700..3288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2780..2799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3459..4250,4251..4319))
                     /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     4082..4101
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4320..4424)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4450..4905)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4587..4608)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4799..4818
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4976..5960
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagc cgctagacat gaatggagct ataggaggtg accttttgct
      121 caattttcct gacatgtcgg tcctagagcg ccaaagggct cacctcaagt acctcaatcc
      181 cacctttgat tctcctctcg ccggcttctt tgccgattct tcaatgatta ccggcggcga
      241 gatggacagc tatctttcga ctgccggttt gaatcttccg atgatgtacg gtgagacgac
      301 ggtggaaggt gattcaagac tctcaatttc gccggaaacg acgcttggga ctggaaattt
      361 caaggcagcg aagtttgata cagagactaa ggattgtaat gaggcggcga agaagatgac
      421 gatgaacaga gatgacctag tagaagaagg agaagaagag aagtcgaaaa taacagagca
      481 aaacaatggg agcacaaaaa gcatcaagaa gatgaaacac aaagccaaga aagaagagaa
      541 caatttctct aatgattcat ctaaagtgac gaaggaattg gagaaaacgg attatattca
      601 tgtaccggtc gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
      661 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
      721 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
      781 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
      841 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
      901 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
      961 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     1021 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     1081 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     1141 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     1201 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     1261 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     1321 caaggctcaa gcttcgaatt ctgcgaagac ctacgattac ctgttcaagc tgctgctgat
     1381 cggggactcg ggggtgggga agacctgtgt cctgttccgc ttctccgagg acgccttcaa
     1441 ctcaactttc atctccacca taggaattga ctttaaaatt aggaccatag agctcgatgg
     1501 caagagaatt aagctacaga tatgggacac agctggtcaa gaacggtttc ggacgatcac
     1561 aacagcctat tacaggggcg caatgggcat catgctggtc tatgacatca ccaacgagaa
     1621 atcctttgac aatatccgga actggattcg gaacattgag gagcatgctt ctgcagatgt
     1681 cgaaaagatg atactcggaa acaagtgtga tgtgaacgac aaaagacaag tttccaagga
     1741 acggggagaa aagctggccc tggactatgg aatcaagttc atggagacca gtgcgaaggc
     1801 caacatcaat gtggagaacg catttttcac tctcgccaga gacatcaaag caaagatgga
     1861 caaaaaattg gaaggcaaca gtccccaagg gagcaaccag ggagtcaaaa tcacaccaga
     1921 ccagcagaag aggagcagct ttttccgatg tgttcttctg tgatcgccag cacagtctgc
     1981 agtctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     2041 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     2101 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2161 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc
     2221 ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg
     2281 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2341 ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2401 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2461 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2521 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2581 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2641 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2701 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2761 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2821 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2881 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2941 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3001 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3061 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3121 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3181 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3241 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3301 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3361 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3421 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3481 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3541 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3601 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3661 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3721 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3781 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3841 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3901 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3961 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4021 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4081 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4141 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4201 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4261 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4321 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4381 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4441 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     4501 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     4561 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     4621 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     4681 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     4741 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     4801 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     4861 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca
     4921 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga
     4981 ccgccaattc aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct
     5041 gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc
     5101 cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg acctggcact
     5161 gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc
     5221 tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat
     5281 atgccaggat caatatatag gcaatatcca atatggccct atgccaatat ggctattggc
     5341 caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta
     5401 ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat
     5461 accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg
     5521 acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg
     5581 taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc
     5641 aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac
     5701 ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt
     5761 actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa
     5821 tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc
     5881 aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa
     5941 gcaatgctcg tttagggaac
//
