LOCUS       sequence-317745-        6011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express recombinant ChpBK protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6011)
  AUTHORS   Zhang Z, Chen LQ, Zhao YL, Yang CG, Roundtree IA, Zhang Z, Ren J, 
            Xie W, He C, Luo GZ
  TITLE     Single-base mapping of m(6)A by an antibody-independent method.
  JOURNAL   Sci Adv. 2019 Jul 3;5(7):eaax0250. doi: 10.1126/sciadv.aax0250. 
            eCollection 2019 Jul.
  PUBMED    31281898
REFERENCE   2  (bases 1 to 6011)
  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: "Sci Adv."; 
            date: "2019-07-3"; volume: "5(7):eaax0250. doi"; pages: " 
            10.1126/sciadv.aax0250. eCollection 2019 Jul"
COMMENT     Sequence Label: PET-3a-chpBK
FEATURES             Location/Qualifiers
     source          1..6011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             674..691
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1302..1457
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1458..1487
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1541..1559)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1555..1602
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1968..1973,1974..1990,1991..1996))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2023..2041)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2109..2213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2214..2282,2283..3074)
                     /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(2432..2451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3245..3833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3734..3753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3987..4004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4019..4158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4084..4103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4244..4266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4260..4451)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5909..5928
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgcgtatta ccataaaaag atgggggaac agtgcaggta
      121 tggtcattcc caatatcgta atgaaagaac ttaacttaca gccggggcag agcgtggagg
      181 cgcaagtgag caacaatcaa ctgattctga cacccatctc caggcgctac tcgcttgatg
      241 aactgctggc acagtgtgac atgaacgccg cggaacttag cgagcaggat gtctggggta
      301 aatccacccc tgcgggtgac gaaatatggt aaagaaaagt gaatttgaac ggggagacat
      361 tgtgctggtt ggctttgatc cagcaagcgg ccatgaacag caaggtgctg gtcgacctgc
      421 gcttgtgctc tccgttcaag cctttaatca actgggaatg acgctggtgg cccccattac
      481 gcagggcgga aattttgccc gttatgccgg atttagcgtt cctttacatt gcgaagaagg
      541 cgatgtgcac ggcgtggtgc tggtgaatca ggtgcggatg atggatctac acgcccggct
      601 ggcaaagcgt attggtctgg ctgcggatga ggtggtggaa gaggcgttat tacgcttgca
      661 ggcggtggtg gaacatcacc atcatcatca ctaaactagt tgccctaccc gagggcgagt
      721 cggtacgcat cgccgacatc gtgccgggtg cgcggcccaa cagtgacaac gccatcgacc
      781 tgaaagtcct tgaccggcat ggcaatcccg tgctcgccga ccggctgttc cactccggcg
      841 agcatccggt gtacacggtg cgtacggtcg aaggtctgcg tgtgacgggc accgcgaacc
      901 acccgttgtt gtgtttggtc gacgtcgccg gggtgccgac cctgctgtgg aagctgatcg
      961 acgaaatcaa gccgggcgat tacgcggtga ttcaacgcag cgcattcagc gtcgactgtg
     1021 caggttttgc ccgcgggaaa cccgaatttg cgcccacaac ctacacagtc ggcgtccctg
     1081 gactggtgcg tttcttggaa gcacaccacc gagacccgga cgcccaagct atcgccgacg
     1141 agctgaccga cgggcggttc tactacgcga aagtcgccag tgtcaccgac gccggcgtgc
     1201 agccggtgta tagccttcgt gtcgacacgg cagaccacgc gtttatcacg aacgggttcg
     1261 tcagccacgc tactggcctc accggtctga actcaggcct cacgacaaat cctggtgtat
     1321 ccgcttggca ggtcaacaca gcttatactg cgggacaatt ggtcacatat aacggcaaga
     1381 cgtataaatg tttgcagccc cacacctcct tggcaggatg ggaaccatcc aacgttcctg
     1441 ccttgtggca gcttcaacat caccatcatc atcaccatca ccatcattaa ggatccggct
     1501 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1561 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1621 tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag tggctccaag
     1681 tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg
     1741 tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat
     1801 gctgtcggaa tggacgatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     1861 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     1921 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     1981 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     2041 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2101 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2161 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2221 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2281 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2341 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2401 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2461 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2521 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2581 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2641 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2701 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2761 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2821 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2881 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2941 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3001 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3061 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3121 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3181 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3241 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3301 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3361 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3421 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3481 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3541 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3601 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3661 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3721 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3781 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3841 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3901 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3961 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4021 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4081 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4141 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4201 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4261 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4321 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4381 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4441 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4501 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4561 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4621 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4681 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4741 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4801 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4861 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
     4921 caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg
     4981 gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct
     5041 ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg
     5101 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc
     5161 ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga
     5221 tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc
     5281 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc
     5341 cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca
     5401 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     5461 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5521 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5581 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5641 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5701 agggcatcgg tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5761 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5821 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5881 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5941 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     6001 tcccgcgaaa t
//
