LOCUS       sequence-496173-        6192 bp    DNA     circular SYN 05-JAN-2026
DEFINITION  A bacterial expression plasmid encoding ciliary targeting sequence 
            of mammalian Arl13b (amino acids 347-363) with N-terminal His and 
            GFP-tagged.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6192)
  AUTHORS   Mahajan D, Madugula V, Lu L
  TITLE     Rab8 and TNPO1 are ciliary transport adaptors for GTPase Arl13b by 
            interacting with its RVEP motif containing ciliary targeting 
            sequence.
  JOURNAL   J Biol Chem. 2023 May;299(5):104604. doi: 10.1016/j.jbc.2023.104604.
            Epub 2023 Mar 11.
  PUBMED    36907439
REFERENCE   2  (bases 1 to 6192)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 5, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem."; date: "2023-05"; volume: "299(5):104604. doi"; pages: " 
            10.1016/j.jbc.2023.104604. Epub 2023 Mar 11"
COMMENT     Sequence Label: His-13bCTS-GFP
FEATURES             Location/Qualifiers
     source          1..6192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     504..523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     757..774
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    789..928
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(854..873)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1014..1036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1030..1221)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2030..3112)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3074..3093
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3113..3190)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3396..3415
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3503..3522
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3503..3521
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3522..3546
                     /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)."
     RBS             3561..3583
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3594..3611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3621..3638
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3645..3689
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3705..3719
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     regulatory      3813..3822
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3819..3821,3822..3824,3825..4538)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3819..3821,3822..3824,3825..4535)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3864..3885)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4125..4144)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4472..4493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4555..4572
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4625..4643)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4639..4686
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4723..5178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4810..4829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5020..5041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5270..6085)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5270..6085)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5993..6012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6077..6096
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
       61 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      121 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
      181 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      241 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      301 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      361 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      421 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      481 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      541 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      601 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      661 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      721 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      781 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
      841 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
      901 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
      961 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     1021 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     1081 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     1141 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     1201 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     1261 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     1321 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     1381 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     1441 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     1501 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     1561 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     1621 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     1681 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     1741 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     1801 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     1861 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     1921 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     1981 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     2041 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2101 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     2161 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     2221 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     2281 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     2341 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     2401 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     2461 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     2521 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     2581 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     2641 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     2701 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     2761 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     2821 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     2881 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2941 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     3001 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     3061 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     3121 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     3181 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     3241 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     3301 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     3361 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     3421 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     3481 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     3541 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atgcaccatc
     3601 atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa accgctgctg
     3661 ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac gacgacaagg
     3721 ccatggctga tatcggatcc gaattcaaaa ctaagaaact aagaatgaaa aggaaccacc
     3781 gggtagaacc acttaatgcg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc
     3841 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3901 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3961 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     4021 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     4081 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     4141 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     4201 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     4261 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     4321 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     4381 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     4441 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     4501 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcact cgagcaccac
     4561 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     4621 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     4681 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     4741 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4801 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4861 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4921 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4981 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5041 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5101 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5161 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5221 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     5281 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     5341 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     5401 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     5461 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     5521 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     5581 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     5641 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     5701 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     5761 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     5821 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     5881 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     5941 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     6001 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     6061 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     6121 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6181 cccgtagaaa ag
//
