LOCUS       sequence-244936-        4938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4938)
  AUTHORS   Tomishige M, Vale RD
  TITLE     Controlling kinesin by reversible disulfide cross-linking. 
            Identifying the motility-producing conformational change.
  JOURNAL   J Cell Biol. 2000 Nov 27. 151(5):1081-92.
  PUBMED    11086009
REFERENCE   2  (bases 1 to 4938)
  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: "J Cell 
            Biol. 2000 Nov 27. 151(5):1081-92."
COMMENT     Sequence Label: RP hk339-GFP
FEATURES             Location/Qualifiers
     source          1..4938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        100..118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             150..172
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1203..1916
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1203..1916
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1233..1261)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1856..1876
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1917..1934
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1995..2013)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2009..2056
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2101..2119)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2187..2291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2292..2360,2361..3152)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2510..2529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3323..3911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3812..3831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4065..4082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4097..4236
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4162..4181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4322..4344
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4338..4529)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
       61 caggacccaa cgctgcccga gatctcgatc ccgcgaaatt aatacgactc actataggga
      121 gaccacaacg gtttccctct agaaataatt ttgtttaact ttaagaagga gatatacata
      181 tggcggacct ggccgagtgc aacatcaaag tgatgtgtcg cttcagacct ctcaacgagt
      241 ctgaagtgaa ccgcggcgac aagtacatcg ccaagtttca gggagaagac acggtcgtga
      301 tcgcgtccaa gccttatgca tttgatcggg tgttccagtc aagcacatct caagagcaag
      361 tgtataatga ctgtgcaaag aagattgtta aagatgtact tgaaggatat aatggaacaa
      421 tatttgcata cggccaaaca tcctctggga agacacacac aatggagggt aaacttcatg
      481 atccagaagg catgggaatt attccaagaa tagtgcaaga tatttttaat tatatttact
      541 ccatggatga aaatttggaa tttcatatta aggtttcata ttttgaaata tatttggata
      601 agataaggga cctgttagat gtttcaaaga ccaacctttc agttcatgaa gacaaaaacc
      661 gagttcccta tgtaaagggg tgcacagagc gttttgtatg tagtccagat gaagttatgg
      721 ataccataga tgaaggaaaa tccaacagac atgtagcagt tacaaatatg aatgaacata
      781 gctctaggag tcacagtata tttcttatta atgtcaaaca agagaacaca caaacggaac
      841 aaaagctgag tggaaaactt tatctagttg atttagctgg tagtgaaaag gttagtaaaa
      901 ctggagctga aggtgctgtg ctggatgaag ctaaaaacat caacaagtca ctttctgctc
      961 ttggaaatgt tatttctgct ttggctgagg gtagtactta tgttccatat cgagatagta
     1021 aaatgacaag aatccttcaa gattcattag gtggcaactg tagaaccact attgtaattt
     1081 gctgctctcc atcatcatac aatgagtctg aaacaaaatc tacactctta tttggccaaa
     1141 gggccaaaac aattaagaac acagtttgtg tcaatgtgga gttaactgca gaacagggta
     1201 ccatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
     1261 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
     1321 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
     1381 ttgtcactac tctgacttat ggtgttcaat gcttttcaag atacccagat catatgaaac
     1441 agcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
     1501 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
     1561 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca
     1621 aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg
     1681 gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag
     1741 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     1801 acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
     1861 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaacatc
     1921 accatcatca ccattaactc gagcagatcc ggctgctaac aaagcccgaa aggaagctga
     1981 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     2041 cttgaggggt tttttgctga aaggaggaac tatatccgga taattcttga agacgaaagg
     2101 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     2161 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2221 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2281 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2341 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2401 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2461 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2521 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2581 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2641 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2701 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2761 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2821 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2881 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2941 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3001 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3061 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3121 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3181 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3241 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3301 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3361 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3421 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3481 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3541 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3601 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3661 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3721 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3781 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3841 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3901 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3961 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4021 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4081 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4141 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4201 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4261 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4321 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4381 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4441 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4501 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4561 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4621 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4681 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4741 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4801 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4861 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4921 ttctgctaac cagtaagg
//
