LOCUS       sequence-9425-ex        4317 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 4317)
  AUTHORS   Kapust RB, Waugh DS
  TITLE     Controlled intracellular processing of fusion proteins by TEV 
            protease.
  JOURNAL   Protein Expr Purif. 2000 Jul . 19(2):312-8.
  PUBMED    10873547
REFERENCE   2  (bases 1 to 4317)
  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: "Protein 
            Expr Purif. 2000 Jul . 19(2):312-8."
COMMENT     Sequence Label: pKM586
FEATURES             Location/Qualifiers
     source          1..4317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(82..100,101..106,107..123,124..129,130..155)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    82..100
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    107..125
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             165..176
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             186..893
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMDKPE
                     EPFQPVKEATQLMN"
     terminator      929..1015
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1550..2500)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2548..2770
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3262..3356)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3387..4181)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3498..3517)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4108..4127
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc ttcacctcga gtccctatca gtgatagaga ttgacatccc tatcagtgat
      121 agagatactg agcacatcag caggacgcac tgaccgaatt cattaaagag gagaaaggta
      181 ccatgggaga aagcttgttt aaggggccgc gtgattacaa cccgatatcg agcaccattt
      241 gtcatttgac gaatgaatct gatgggcaca caacatcgtt gtatggtatt ggatttgggc
      301 ccttcatcat tacaaacaag cacttgttta gaagaaataa tggaacactg ttggtccaat
      361 cactacatgg tgtattcaag gtcaagaaca ccacgacttt gcaacaacac ctcatcgatg
      421 ggcgggacat gatcattatt cgaatgccta aggatttccc accatttcct caaaagctga
      481 aatttcgcga gccacaacgg gaagagcgca tttgtcttgt gacaaccaac ttccaaacta
      541 agagcatgtc tagcatggtg tcagacacta gttgcacatt cccttcatct gatggcatat
      601 tctggaagca ttggattcaa accaaggatg ggcagtgtgg cagtccatta gtatcaactc
      661 gagatgggtt cattgttggt atacactcag catcgaattt caccaacaca aacaattatt
      721 tcacaagcgt gccgaaaaac ttcatggaat tgttgacaaa tcaggaggcg cagcagtggg
      781 ttagtggttg gcgattaaat gctgactcag tattgtgggg gggccataaa gttttcatgg
      841 acaaacctga agagcctttt cagccagtta aggaagcgac tcaactcatg aattaataag
      901 gatcccatgg tacgcgtgct agaggcatca aataaaacga aaggctcagt cgaaagactg
      961 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     1021 gccctagacc tagggtacgg gttttgctgc ccgcaaacgg gctgttctgg tgttgctagt
     1081 ttgttatcag aatcgcagat ccggcttcag gtttgccggc tgaaagcgct atttcttcca
     1141 gaattgccat gattttttcc ccacgggagg cgtcactggc tcccgtgttg tcggcagctt
     1201 tgattcgata agcagcatcg cctgtttcag gctgtctatg tgtgactgtt gagctgtaac
     1261 aagttgtctc aggtgttcaa tttcatgttc tagttgcttt gttttactgg tttcacctgt
     1321 tctattaggt gttacatgct gttcatctgt tacattgtcg atctgttcat ggtgaacagc
     1381 tttaaatgca ccaaaaactc gtaaaagctc tgatgtatct atctttttta caccgttttc
     1441 atctgtgcat atggacagtt ttccctttga tatctaacgg tgaacagttg ttctactttt
     1501 gtttgttagt cttgatgctt cactgataga tacaagagcc ataagaacct cagatccttc
     1561 cgtatttagc cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg
     1621 aaccattgag atcatgctta ctttgcatgt cactcaaaaa ttttgcctca aaactggtga
     1681 gctgaatttt tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta cgtaggtagg
     1741 aatctgatgt aatggttgtt ggtattttgt caccattcat ttttatctgg ttgttctcaa
     1801 gttcggttac gagatccatt tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg
     1861 ggcggcctcg cttatcaacc accaatttca tattgctgta agtgtttaaa tctttactta
     1921 ttggtttcaa aacccattgg ttaagccttt taaactcatg gtagttattt tcaagcatta
     1981 acatgaactt aaattcatca aggctaatct ctatatttgc cttgtgagtt ttcttttgtg
     2041 ttagttcttt taataaccac tcataaatcc tcatagagta tttgttttca aaagacttaa
     2101 catgttccag attatatttt atgaattttt ttaactggaa aagataaggc aatatctctt
     2161 cactaaaaac taattctaat ttttcgcttg agaacttggc atagtttgtc cactggaaaa
     2221 tctcaaagcc tttaaccaaa ggattcctga tttccacagt tctcgtcatc agctctctgg
     2281 ttgctttagc taatacacca taagcatttt ccctactgat gttcatcatc tgagcgtatt
     2341 ggttataagt gaacgatacc gtccgttctt tccttgtagg gttttcaatc gtggggttga
     2401 gtagtgccac acagcataaa attagcttgg tttcatgctc cgttaagtca tagcgactaa
     2461 tcgctagttc atttgctttg aaaacaacta attcagacat acatctcaat tggtctaggt
     2521 gattttaatc actataccaa ttgagatggg ctagtcaatg ataattacta gtccttttcc
     2581 cgggagatct gggtatctgt aaattctgct agacctttgc tggaaaactt gtaaattctg
     2641 ctagaccctc tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat attcaagtgg
     2701 ttataattta tagaataaag aaagaataaa aaaagataaa aagaatagat cccagccctg
     2761 tgtataactc actactttag tcagttccgc agtattacaa aaggatgtcg caaacgctgt
     2821 ttgctcctct acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct
     2881 cgggcaaatc gctgaatatt ccttttgtct ccgaccatca ggcacctgag tcgctgtctt
     2941 tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag tgaatggggg taaatggcac
     3001 tacaggcgcc ttttatggat tcatgcaagg aaactaccca taatacaaga aaagcccgtc
     3061 acgggcttct cagggcgttt tatggcgggt ctgctatgtg gtgctatctg actttttgct
     3121 gttcagcagt tcctgccctc tgattttcca gtctgaccac ttcggattat cccgtgacag
     3181 gtcattcaga ctggctaatg cacccagtaa ggcagcggta tcatcaacag gcttacccgt
     3241 cttactgtcc ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
     3301 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
     3361 cgagctctcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     3421 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     3481 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     3541 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     3601 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     3661 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     3721 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     3781 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     3841 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     3901 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     3961 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     4021 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     4081 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     4141 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     4201 gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc
     4261 cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattcc
//
