LOCUS       sequence-456189-        7822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in BL21(DE3) of T4 primase I342G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7822)
  AUTHORS   Valentine AM, Ishmael FT, Shier VK, Benkovic SJ
  TITLE     A zinc ribbon protein in DNA replication: primer synthesis and 
            macromolecular interactions by the bacteriophage T4 primase.
  JOURNAL   Biochemistry. 2001 Dec 18;40(50):15074-85. doi: 10.1021/bi0108554.
  PUBMED    11735390
REFERENCE   2  (bases 1 to 7822)
  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"; doi: "10"; journalName: 
            "Biochemistry"; date: "2001-12-18- 18"; volume: "40"; issue: "50"; 
            pages: "15074-85"
COMMENT     Sequence Label: pET-Impact-T4gp61(I342G)
FEATURES             Location/Qualifiers
     source          1..7822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(9..20)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(476..1294)
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     RBS             2328..2350
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2365..2389
                     /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)."
     primer_bind     complement(2389..2408)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2390..2408)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2492..2511)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2717..2794
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2795..3877
                     /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"
     primer_bind     complement(2814..2833)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4686..4877
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4871..4893)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4979..5118
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5034..5053
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5133..5150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5304..5892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5384..5403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6003..6022)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6014..6829
                     /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             6014..6829
                     /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     complement(6087..6106)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6921..7376)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7058..7079)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7270..7289
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(7413..7460)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7456..7474
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(7527..7544)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(7566..7721)
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
ORIGIN
        1 aataatcgtc ttccttcaat tcttgtaact cgaaataaaa tccgcggcac tcacgtgcaa
       61 aagcagcggc gggagcaggc ctgaattttt tagagccggc acacttcgaa agaacgttaa
      121 gcaaaacatc tccaccagac atataaatag cataactaat tttatgtttg gtgacattca
      181 tgtcaacctt agcaggttct gcgttaaccg agactactaa gcctaaagaa cgagcaaggg
      241 aaaccaaacc atctctgaca gaagtatgaa ttgtctttat tgttgcttta ataccatgct
      301 catcagtaac atagccatca gaatcaatta taccagcaag aaatgtttca cgagtaccga
      361 tattgtccgt agacaagaaa gaaggaatat ttttgacacc gtccttcaag aatcctaagc
      421 caacaatagc gtcccataat ggattctcag tattaagatt attgcgaata ccattacctc
      481 tgacaacttt agagtacaaa ttaacagttt tggcaacttg tggttctttt ctgtccttat
      541 actcggcgca caaattcaac ttttcagcat attcagtaac acgttccatc aaagaagtat
      601 ctctggaatc aaccgaaaaa gttgccctgt cagacaatcc atcaccaatc cataaaccaa
      661 gtaaataagc aagtactttt ggaccttcaa tggtgagatg aaacttactt ttttgcatgt
      721 agtcgaaaaa gtggtcattc tcataaagaa ttggagcgta agtctggtag gtagctttac
      781 gaacatggga acccaacaga gaaagatctc tggcctcaat agtccactca aaataagctt
      841 tatttgaagc ctttctatag gattctacta attcgttggc tctctcaggc ccctcagata
      901 ttgggtagct ctttgaaact tccttgacaa gctcaacaat tctaccgtcg ggggctttct
      961 tttggcccat ctcaaaagta ataacttcaa aatattcgac acccttaatg gtacgagaca
     1021 aacggcggac actacgaggt gttctaacaa ccaactcatg ggtcgcatta cacgtaaact
     1081 tgagtaattc tggcacttca cgacttgagt cacttttgtg ggctctgtgc tgacttttct
     1141 gcacgacgct gtacatagtt tctcttcctc tgggcaattt aattacctca cgaggtctgc
     1201 catctttacc catgacctta ttaccaacct caatgttttc aatacattca atagacccat
     1261 ccgccattaa aacattggta cccttggcaa agcaaccctt agcatattta gatagccgca
     1321 ttttagccat taacccctgg gcaatattat ttttcatata ttccataatt tgttcagggg
     1381 ttgcaccttc ctttctaatc atatcattaa catcttttga tttccaggga gatttatccc
     1441 aaaacataac cctttctcct gcatcaacta atttagtcat tcgtttaata gtgtcagggt
     1501 gacgaggttc attatctaag acccacacac gtctatcttt aaatggaaca acttctaggt
     1561 ctaattgacc gcccgtaata gctataccat tttcaataaa aagtgaatct ataggtcctt
     1621 ctagaacata tacatcacca tctttaactc gttcgactcc atagattttt gttgcctcag
     1681 gataagcttt gatggtgata tatttttgag gagcatcttt ctttaatgca cgtccttgaa
     1741 aagactcagc ttttccatta gcattataaa ttggaataac aagacgaggc tcagaaattt
     1801 cctttttgta tgttcccggt gctatgctat taactaattt aggccattcg gttgtaaacc
     1861 aaagatattt ccatttatcc tttggaatac aacgagcttt tacgtatttt ataattggat
     1921 ggtcttccgc cagtttatct aatctaacac atgacggaag agatttaatt attttcttct
     1981 cgggttgttt aggaagttct ttaggttttt ctattggacg acttttacct ttttcttttc
     2041 ttatttcaaa gatatactca cgatataaat cgggttcaaa ctcctttaaa tatattccga
     2101 ttggtgcatg atagttacag ttataacaat gaatatttcc ttcattatta tcaccataat
     2161 accatccacg ggctttattc tggtcggttt ttgaatctcc acaaacaggg catctaaacc
     2221 gtaatttaaa agttgaacta ttatttactt gtgtgaattt aggtaaatga gctaatgcac
     2281 ggtatgcaaa ctcattatca atccaaggta ttgatgacat atgtatatct ccttcttaaa
     2341 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     2401 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
     2461 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     2521 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     2581 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     2641 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     2701 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     2761 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     2821 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     2881 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     2941 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     3001 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     3061 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     3121 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     3181 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     3241 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     3301 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     3361 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     3421 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     3481 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     3541 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     3601 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     3661 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     3721 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     3781 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3841 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     3901 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     3961 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     4021 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     4081 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     4141 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     4201 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     4261 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     4321 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     4381 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     4441 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     4501 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     4561 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     4621 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     4681 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     4741 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     4801 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     4861 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4921 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4981 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     5041 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     5101 agaaaatacc gcatcaggcg ctcccccgct tcctcgctca ctgactcgct gcgctcggtc
     5161 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5221 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5281 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5341 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5401 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5461 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5521 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5581 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5641 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5701 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5761 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5821 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5881 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5941 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     6001 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     6061 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     6121 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     6181 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     6241 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     6301 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     6361 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     6421 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     6481 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     6541 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     6601 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     6661 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     6721 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     6781 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     6841 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6901 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     6961 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     7021 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     7081 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     7141 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     7201 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     7261 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     7321 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     7381 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     7441 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     7501 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag tgcggccgct
     7561 tatcattgaa gctgccacaa ggcaggaacg ttggatggtt cccatcctgc caaggaggtg
     7621 tggggctgca aacatttata cgtcttgccg ttatatgtga ccaattgtcc cgcagtataa
     7681 gctgtgttga cctgccaagc ggatacacca ggatttgtcg tgaggcctga gttcagaccg
     7741 gtcaggccac cgcatgcatg gacgacaacc tgggatccaa gcaaaaactg atgatcagaa
     7801 tcatcagata aagtaatccc at
//
