LOCUS       sequence-355089-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human KIF1A(1-393) fused with leucine zipper, mScarlet and
            StrepII tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7245)
  AUTHORS   Anazawa Y, Kita T, Iguchi R, Hayashi K, Niwa S
  TITLE     De novo mutations in KIF1A-associated neuronal disorder (KAND) 
            dominant-negatively inhibit motor activity and axonal transport of 
            synaptic vesicle precursors.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Aug 9;119(32):e2113795119. doi: 
            10.1073/pnas.2113795119. Epub 2022 Aug 2.
  PUBMED    35917346
REFERENCE   2  (bases 1 to 7245)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-08-9"; volume: "119(32):e2113795119. 
            doi"; pages: " 10.1073/pnas.2113795119. Epub 2022 Aug 2"
COMMENT     Sequence Label: pSN554
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2764..3456
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(2894..2912)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3162..3181
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3463..3486
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3490..3507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3560..3578)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3574..3621
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3658..4113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3745..3764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3955..3976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4205..5020)
                     /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(4205..5020)
                     /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     4928..4947
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5012..5031
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5142..5730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5631..5650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5884..5901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5916..6055
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5981..6000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6141..6163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6157..6348)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7157..7245,1..994))
                     /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"
ORIGIN
        1 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atataccatg ggcagcagca tggccggggc ttcggtgaag
     1501 gtggcggtgc gggtccgccc cttcaattcc cgggaaatga gccgtgactc caagtgcatc
     1561 attcagatgt ctggaagcac caccaccatt gttaacccca aacagcccaa ggagacgccc
     1621 aaaagcttca gctttgacta ctcctactgg tcgcacacct cacctgagga catcaactac
     1681 gcgtcgcaga agcaggtgta ccgggacatc ggcgaggaga tgctgcagca tgcctttgag
     1741 ggatacaacg tgtgcatctt cgcctatggg cagacgggtg ccggcaagtc ctacaccatg
     1801 atgggcaagc aggagaagga ccagcagggc atcatcccac agctctgcga ggacctcttc
     1861 tctcggatca acgacacgac caacgacaac atgtcctact ccgtggaggt cagctacatg
     1921 gagatttact gtgagcgcgt ccgtgacctc ctgaacccca agaacaaggg caaccttcgc
     1981 gtgagggagc acccactgct ggggccctac gtggaggacc tctccaagct ggctgtcacc
     2041 tcctacaatg acatccagga cctcatggac tcagggaaca aggccaggac cgtggcggcc
     2101 accaacatga atgagaccag cagtcgctcc cacgccgtct tcaacatcat cttcacccag
     2161 aagcgccatg acgcagagac caatatcacc acggagaagg tgagcaaaat cagcctggtg
     2221 gacctggctg ggagcgagcg ggctgactcc acgggagcca agggcacgcg cctcaaggag
     2281 ggggccaaca tcaacaagtc gctgaccacc ctgggcaagg tcatctccgc cctggctgaa
     2341 atggactccg gacccaacaa gaacaagaaa aagaagaaga cagatttcat tccgtaccga
     2401 gattccgtgt tgacctggct cctccgggaa aacctgggcg gtaactcaag gacagctatg
     2461 gtggcagcct tgagtcctgc agacatcaac tacgatgaga cccttagcac gctgaggtat
     2521 gctgaccggg ccaagcagat ccgctgcaat gctgtcatca atgaggaccc caacaacaag
     2581 ctgatccgcg agctgaagga tgaggtgacc cggctgcggg accttctgta cgcccagggt
     2641 cttggcgaca tcactgacgg cgcgggagtg aaacaacttg aagacaaggt tgaagaattg
     2701 gcttcgaaaa attatcactt ggaaaatgag gttgccagat taaagaaatt agttgcggcc
     2761 gcagtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
     2821 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     2881 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
     2941 atcctgtccc ctcagttcat gtacggctcc agggccttca ccaagcaccc cgccgacatc
     3001 cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     3061 gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg caccctgatc
     3121 tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat gcagaagaag
     3181 acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt gctgaagggc
     3241 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
     3301 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg
     3361 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgagggc
     3421 cgccactcca ccggcggcat ggacgagctg tacaagctcg agtggagtca tccacaattt
     3481 gagaagtaac accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     3541 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     3601 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     3661 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3721 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3781 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3841 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3901 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3961 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4021 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4081 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     4141 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     4201 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     4261 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     4321 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     4381 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     4441 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     4501 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     4561 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     4621 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     4681 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     4741 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     4801 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     4861 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     4921 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     4981 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     5041 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     5101 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5161 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5221 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5281 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5341 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5401 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5461 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5521 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5581 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5641 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5701 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5761 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5821 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5881 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     5941 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     6001 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     6061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6181 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     6241 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     6301 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     6361 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     6421 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     6481 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     6541 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     6601 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     6661 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     6721 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     6781 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     6841 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     6901 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     6961 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     7021 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     7081 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     7141 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7201 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgg
//
