LOCUS       sequence-426157-        5328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with C terminal non cleavable His10 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5328)
  AUTHORS   Fairhead M, Koekemoer L, Williams E, von Delft F
  TITLE     A selection of Golden Gate vectors to simplify recombinant protein 
            production in Escherichia coli
  JOURNAL   bioRxiv 2024.02.13.579886
REFERENCE   2  (bases 1 to 5328)
  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: "bioRxiv 
            2024.02.13.579886"
COMMENT     Sequence Label: pNIC-CH10-GG
FEATURES             Location/Qualifiers
     source          1..5328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..56
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        37..55
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    56..80
                     /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)."
     CDS             160..189
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             229..246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(299..317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      313..360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      397..852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(484..503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     694..715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(944..1759)
                     /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             complement(944..1759)
                     /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     1667..1686
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1751..1770
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1881..2469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2370..2389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2623..2640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2655..2794
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2720..2739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2880..2902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2896..3087)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3896..4978)
                     /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     4940..4959
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4979..5056)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5262..5281
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
       61 tgtgagcgga taacaattcc cctctagaaa taattttgtt taaccttaag aaggagatat
      121 acacatgaga gaccgcggat aattcgggtc tcactcggac atcatcatca ccaccaccat
      181 catcaccact aatgagcacc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
      241 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      301 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      361 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      421 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      481 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      541 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      601 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      661 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      721 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      781 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      841 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      901 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
      961 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1021 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1081 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1141 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1201 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1261 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1321 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1381 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1441 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     1501 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     1561 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     1621 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     1681 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     1741 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     1801 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1861 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1921 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1981 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2041 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2101 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2161 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2221 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2281 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2341 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2401 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2461 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2521 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2581 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2641 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2701 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     2761 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2821 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2881 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     2941 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3001 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3061 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3121 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3181 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3241 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3301 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3361 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3421 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3481 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3541 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     3601 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     3661 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     3721 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     3781 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     3841 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3901 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3961 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4021 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4081 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4141 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4201 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4261 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4321 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4381 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4441 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4501 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4561 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     4621 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     4681 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     4741 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     4801 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     4861 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4921 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4981 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5041 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5101 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5161 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5221 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     5281 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgt
//
