LOCUS       sequence-429348-        5663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 SUMO2 tag and SENP1 
            cleavage non cleavable N-terminal avitag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5663)
  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 5663)
  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-HIS6-SUMO1-AVITAG-GG
FEATURES             Location/Qualifiers
     source          1..5663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    60..84
                     /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             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             132..149
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             568..585
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(638..656)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      652..699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      736..1191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(823..842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1033..1054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1283..2098)
                     /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(1283..2098)
                     /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     2006..2025
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2090..2109
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2220..2808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2709..2728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2962..2979
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2994..3133
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3059..3078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3219..3241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3235..3426)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4235..5317)
                     /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     5279..5298
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5318..5395)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5601..5620
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
       61 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      121 atatacatat gcaccaccat catcaccatg gctcaggaga tcaggaagcg aaaccttcga
      181 ccgaggacct gggcgacaag aaagaagggg agtatattaa gttaaaggtg atcggccagg
      241 actcaagcga gatccacttc aaagtcaaaa tgaccacgca cctcaagaag ttgaaggaga
      301 gttactgtca acggcaaggg gttcctatga actcgcttcg cttcctgttc gagggccagc
      361 gaattgcgga caaccacaca cctaaggaac ttggtatgga agaggaagac gtcattgagg
      421 tgtaccagga gcagacaggc ggcggatctc tgaacgatat ttttgaagcg cagaaaattg
      481 aatggcatga atctggttcc atgagagacc gcggataatt cgggtctcac tcgagcaccc
      541 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
      601 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      661 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      721 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      781 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      841 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      901 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      961 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1021 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1081 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1141 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1201 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1261 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1321 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     1381 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     1441 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     1501 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     1561 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     1621 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     1681 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1741 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1801 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1861 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     1921 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     1981 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2041 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2101 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2161 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2221 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2281 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2341 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2401 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2461 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2521 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2581 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2641 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2701 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2761 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2821 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2881 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2941 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3001 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3061 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3121 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3181 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3241 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3301 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3361 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3421 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     3481 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     3541 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3601 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     3661 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3721 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3781 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3841 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3901 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3961 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4021 gctccagcga aagtggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4081 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4141 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4201 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4261 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4321 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4381 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4441 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4501 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4561 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4621 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4681 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4741 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4801 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4861 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4921 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4981 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5041 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5101 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5161 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5221 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5281 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5341 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5401 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5461 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5521 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5581 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5641 gcgccggtga tgccggccac gat
//
