LOCUS       sequence-426140-        5486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal PelB SEC pathway signal 
            peptide His10 and AVITAG and TEV cleavage .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5486)
  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 5486)
  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-PelB-NH10T-AVITAG-GG
FEATURES             Location/Qualifiers
     source          1..5486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     118..183
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             193..222
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             289..309
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             380..397
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(450..468)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      464..511
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      548..1003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(635..654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     845..866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1095..1910)
                     /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(1095..1910)
                     /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     1818..1837
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1902..1921
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2032..2620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2521..2540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2774..2791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2806..2945
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2871..2890)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3031..3053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3047..3238)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4047..5129)
                     /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     5091..5110
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5130..5207)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5413..5432
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      121 aaatacctgc tgccgaccgc tgctgctggt ctgctgcttc tcgctgccca gccggcgatg
      181 gcctcttctg gtcatcacca tcaccatcac catcaccatc acggttctgg atctctgaac
      241 gatatttttg aagcgcagaa aattgaatgg catgaatctg gtggtggtga gaacctgtac
      301 ttccaatcct ccatgagaga ccgcggataa ttcgggtctc actcgagcac ccgtcgacaa
      361 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      421 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      481 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
      541 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      601 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      661 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      721 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      781 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      841 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      901 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      961 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1021 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1081 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     1141 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     1201 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     1261 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     1321 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     1381 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     1441 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     1501 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     1561 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     1621 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     1681 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     1741 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     1801 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     1861 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     1921 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     1981 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2041 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2101 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2161 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2221 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2281 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2341 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2401 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2461 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2521 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2581 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2641 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2701 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2761 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2821 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2881 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2941 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3001 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3061 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3121 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3181 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3241 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3301 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3361 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3421 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3481 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3541 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3601 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3661 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3721 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3781 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3841 gaaagtggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3901 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3961 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4021 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4081 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4141 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4201 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4261 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4321 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4381 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4441 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4501 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4561 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4621 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4681 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4741 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4801 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4861 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4921 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4981 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5041 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5101 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5161 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5221 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5281 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5341 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5401 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5461 gatgccggcc acgatgcgtc cggcgt
//
