LOCUS       sequence-334719-        5409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express in mammalian cells a target protein C-terminally fused to 
            the Mxe GryA intein - chitin binding domain (CBD) tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5409)
  AUTHORS   Carman PJ, Barrie KR, Dominguez R
  TITLE     Novel human cell expression method reveals the role and prevalence 
            of posttranslational modification in nonmuscle tropomyosins.
  JOURNAL   J Biol Chem. 2021 Oct;297(4):101154. doi: 10.1016/j.jbc.2021.101154.
            Epub 2021 Sep 1.
  PUBMED    34478714
REFERENCE   2  (bases 1 to 5409)
  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 Biol 
            Chem."; date: "2021-10"; volume: "297(4):101154. doi"; pages: " 
            10.1016/j.jbc.2021.101154. Epub 2021 Sep 1"
COMMENT     Sequence Label: pJCX4
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             275..868
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             899..1054
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(1119..1137)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1133..1180
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    1228..1816
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1281..1301)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1699..1710)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    1833..1954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1870..1889)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1924..1943
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1961..2416)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2098..2119)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2310..2329
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2443..2547
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2549..2906
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2549..2740
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2572..2592)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2757..2892
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2941..3735
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2941..3735
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2995..3014)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3605..3624
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3923..3942)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3967..4014
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4343..4931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4832..4851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5106..5409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gctcgcgagt
      241 cgacggcggc cgcgaattcc tcgagggctc ttcctgcatc acgggagatg cactagttgc
      301 cctacccgag ggcgagtcgg tacgcatcgc cgacatcgtg ccgggtgcgc ggcccaacag
      361 tgacaacgcc atcgacctga aagtccttga ccggcatggc aatcccgtgc tcgccgaccg
      421 gctgttccac tccggcgagc atccggtgta cacggtgcgt acggtcgaag gtctgcgtgt
      481 gacgggcacc gcgaaccacc cgttgttgtg tttggtcgac gtcgccgggg tgccgaccct
      541 gctgtggaag ctgatcgacg aaatcaagcc gggcgattac gcggtgattc aacgcagcgc
      601 attcagcgtc gactgtgcag gttttgcccg cgggaaaccc gaatttgcgc ccacaaccta
      661 cacagtcggc gtccctggac tggtgcgttt cttggaagca caccaccgag acccggacgc
      721 ccaagctatc gccgacgagc tgaccgacgg gcggttctac tacgcgaaag tcgccagtgt
      781 caccgacgcc ggcgtgcagc cggtgtatag ccttcgtgtc gacacggcag accacgcgtt
      841 tatcacgaac gggttcgtca gccacgctac tggcctcacc ggtctgaact caggcctcac
      901 gacaaatcct ggtgtatccg cttggcaggt caacacagct tatactgcgg gacaattggt
      961 cacatataac ggcaagacgt ataaatgttt gcagccccac acctccttgg caggatggga
     1021 accatccaac gttcctgcct tgtggcagct tcaatgactg caggaagggg atccggctgc
     1081 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1141 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1201 cggaggagac aataccggaa tctagataat caacctctgg attacaaaat ttgtgaaaga
     1261 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     1321 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     1381 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     1441 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     1501 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     1561 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     1621 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     1681 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     1741 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     1801 tgggccgcct ccccgcatca attgttgttg ttaacttgtt tattgcagct tataatggtt
     1861 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1921 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     1981 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2041 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2101 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2161 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2221 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2281 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2341 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2401 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2461 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2521 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     2581 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2641 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2701 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     2761 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     2821 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     2881 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     2941 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3001 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3061 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3121 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3181 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3241 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3301 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3361 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgag
     3421 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3481 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3541 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3601 atagcgttgg ctacccgtga tattgctgag gagcttggcg gcgaatgggc tgaccgcttc
     3661 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3721 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3781 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3841 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3901 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     3961 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4021 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4081 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4141 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4201 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4261 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4321 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4381 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4441 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4501 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4561 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4621 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4681 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4741 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4801 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4861 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4921 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4981 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     5041 tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     5101 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     5161 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     5221 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     5281 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     5341 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     5401 attaccatg
//
