LOCUS       sequence-382140-        5949 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For use in light-induced Rab35 protein inactivation through the 
            interaction with CRY2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5949)
  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 5949)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-mRuby-Rab35
FEATURES             Location/Qualifiers
     source          1..5949
                     /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             101..610
                     /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      629..638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             635..1345
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     polyA_signal    2001..2135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2025..2044)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2079..2098
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2248..2268)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2248..2266)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2287..2303)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2287..2303)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2300..2322)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2311..2327
                     /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(2335..2341,2342..2359,2360..2365))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2380..2401
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2518..2535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2689..3277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2769..2788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3448..4239,4240..4308))
                     /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     4071..4090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4309..4413)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4439..4894)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4576..4597)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4788..4807
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4965..5949
                     /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 tttttgcagg tcagatccgc tagcgccacc atgaatggag ctataggagg
      121 tgaccttttg ctcaattttc ctgacatgtc ggtcctagag cgccaaaggg ctcacctcaa
      181 gtacctcaat cccacctttg attctcctct cgccggcttc tttgccgatt cttcaatgat
      241 taccggcggc gagatggaca gctatctttc gactgccggt ttgaatcttc cgatgatgta
      301 cggtgagacg acggtggaag gtgattcaag actctcaatt tcgccggaaa cgacgcttgg
      361 gactggaaat ttcaaggcag cgaagtttga tacagagact aaggattgta atgaggcggc
      421 gaagaagatg acgatgaaca gagatgacct agtagaagaa ggagaagaag agaagtcgaa
      481 aataacagag caaaacaatg ggagcacaaa aagcatcaag aagatgaaac acaaagccaa
      541 gaaagaagag aacaatttct ctaatgattc atctaaagtg acgaaggaat tggagaaaac
      601 ggattatatt catgtaccgg tcgccacagc caccatggtg tctaagggcg aagagctgat
      661 caaggaaaat atgcgtatga aggtggtcat ggaaggttcg gtcaacggcc accaattcaa
      721 atgcacaggt gaaggagaag gcaatccgta catgggaact caaaccatga ggatcaaagt
      781 catcgaggga ggacccctgc catttgcctt tgacattctt gccacgtcgt tcatgtatgg
      841 cagccgtact tttatcaagt acccgaaagg cattcctgat ttctttaaac agtcctttcc
      901 tgagggtttt acttgggaaa gagttacgag atacgaagat ggtggagtcg tcaccgtcat
      961 gcaggacacc agccttgagg atggctgtct cgtttaccac gtccaagtca gaggggtaaa
     1021 ctttccctcc aatggtcccg tgatgcagaa gaagaccaag ggttgggagc ctaatacaga
     1081 gatgatgtat ccagcagatg gtggtctgag gggatacact catatggcac tgaaagttga
     1141 tggtggtggc catctgtctt gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg
     1201 gaacatcaag atgcccggta tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag
     1261 tgacaatgaa atgttcgtag tacaacgcga acacgcagtt gccaagttcg ccgggcttgg
     1321 tggtgggatg gacgagctgt acaagcgagc tcaagcttcg atggcccggg actacgacca
     1381 cctcttcaag ctgctcatca tcggcgacag cggtgtgggc aagagcagtt tactgttgcg
     1441 ttttgcagac aacactttct caggcagcta catcaccacg atcggagtgg atttcaagat
     1501 ccggaccgtg gagatcaacg gggagaaggt gaagctgcag atctgggaca cagcggggca
     1561 ggagcgcttc cgcaccatca cctccacgta ttatcggggg acccacgggg tcattgtggt
     1621 ttacgacgtc accagtgccg agtcctttgt caacgtcaag cggtggcttc acgaaatcaa
     1681 ccagaactgt gatgatgtgt gccgaatatt agtgggtaat aagaatgacg accctgagcg
     1741 gaaggtggtg gagacggaag atgcctacaa attcgccggg cagatgggca tccagttgtt
     1801 cgagaccagc gccaaggaga atgtcaacgt ggaagagatg ttcaactgca tcacggagct
     1861 ggtcctccga gcaaagaaag acaacctggc aaaacagcag cagcaacaac agaacgatgt
     1921 ggtgaagctc acgaagaaca gtaaacgaaa gaaacgctgc tgctaatgca gtctagaact
     1981 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     2041 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     2101 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     2161 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc
     2221 attgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     2281 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2341 acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2401 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2461 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2521 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2581 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2641 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2701 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2761 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2821 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2881 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2941 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3001 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3061 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4381 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     4441 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     4501 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     4561 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     4621 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     4681 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     4741 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     4801 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4861 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     4921 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagtcgac cgccaattca
     4981 atatggcgta tatggactca tgccaattca atatggtgga tctggacctg tgccaattca
     5041 atatggcgta tatggactcg tgccaattca atatggtgga tctggacccc agccaattca
     5101 atatggcgga cttggcacca tgccaattca atatggcgga cctggcactg tgccaactgg
     5161 ggaggggtct acttggcacg gtgccaagtt tgaggagggg tcttggccct gtgccaagtc
     5221 cgccatattg aattggcatg gtgccaataa tggcggccat attggctata tgccaggatc
     5281 aatatatagg caatatccaa tatggcccta tgccaatatg gctattggcc aggttcaata
     5341 ctatgtattg gccctatgcc atatagtatt ccatatatgg gttttcctat tgacgtagat
     5401 agcccctccc aatgggcggt cccatatacc atatatgggg cttcctaata ccgcccatag
     5461 ccactccccc attgacgtca atggtctcta tatatggtct ttcctattga cgtcatatgg
     5521 gcggtcctat tgacgtatat ggcgcctccc ccattgacgt caattacggt aaatggcccg
     5581 cctggctcaa tgcccattga cgtcaatagg accacccacc attgacgtca atgggatggc
     5641 tcattgccca ttcatatccg ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc
     5701 cacttggcag tacatcaata tctattaata gtaacttggc aagtacatta ctattggaag
     5761 tacgccaggg tacattggca gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat
     5821 ggctgccaag tacatcccca ttgacgtcaa tggggagggg caatgacgca aatgggcgtt
     5881 ccattgacgt aaatgggcgg taggcgtgcc taatgggagg tctatataag caatgctcgt
     5941 ttagggaac
//
