LOCUS       sequence-426314-        5330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal non cleavable His10 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5330)
  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 5330)
  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-NH10-GG
FEATURES             Location/Qualifiers
     source          1..5330
                     /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)."
     RBS             95..117
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             128..157
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             231..248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(301..319)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      315..362
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      399..854
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(486..505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     696..717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(946..1761)
                     /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(946..1761)
                     /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     1669..1688
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1753..1772
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1883..2471
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2372..2391
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2625..2642
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2657..2796
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2722..2741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2882..2904
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2898..3089)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3898..4980)
                     /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     4942..4961
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4981..5058)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5264..5283
                     /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 taactttaag aaggagatat
      121 acatatgcat catcatcacc accaccatca tcatcacggt tccatgagag accgcggata
      181 attcgggtct cactcgagca cccgtcgaca agcttgcggc cgcactcgag caccaccacc
      241 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      301 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      361 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      421 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      481 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      541 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      601 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      661 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      721 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      781 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
      841 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      901 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
      961 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1021 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1081 ctggtatcgg tctgctattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1141 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1201 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1261 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1321 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1381 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1441 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1501 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1561 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1621 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1681 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     1741 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     1801 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1861 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1921 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1981 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2041 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2101 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2161 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2221 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2281 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2341 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2401 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2461 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2521 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2581 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2641 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2701 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     2761 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     2821 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2881 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     2941 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3001 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3061 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3121 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3181 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3241 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3301 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3361 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3421 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3481 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3541 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     3601 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     3661 agcgacaggc cgatcatcgt cgcgctccag cgaaagtggt cctcgccgaa aatgacccag
     3721 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     3781 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     3841 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     3901 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3961 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4021 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4081 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4141 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4201 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4261 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4321 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4381 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4441 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4501 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4561 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4621 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4681 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4741 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4801 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4861 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     4921 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     4981 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5041 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5101 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5161 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5221 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5281 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
//
