LOCUS       sequence-426159-        5291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with no tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5291)
  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 5291)
  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-NoTag-GG
FEATURES             Location/Qualifiers
     source          1..5291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             176..193
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(246..264)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      260..307
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      344..799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(431..450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     641..662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(891..1706)
                     /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(891..1706)
                     /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     1614..1633
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1698..1717
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1828..2416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2317..2336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2570..2587
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2602..2741
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2667..2686)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2827..2849
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2843..3034)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3843..4925)
                     /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     4887..4906
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4926..5003)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5209..5228
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaacct taagaaggag atatacacat gagagaccgc
      121 ggataattcg ggtctcactc gagcacccgt cgacaagctt gcggccgcac tcgagcacca
      181 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
      241 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
      301 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
      361 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      421 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      481 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      541 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      601 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      661 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
      721 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      781 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      841 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
      901 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
      961 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     1021 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     1081 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     1141 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     1201 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     1261 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     1321 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     1381 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     1441 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     1501 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     1561 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     1621 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     1681 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     1741 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1801 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1861 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1921 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     1981 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2041 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2101 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2161 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2221 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2281 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2341 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2401 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2461 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2521 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2581 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2641 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     2701 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     2761 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     2821 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     2881 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     2941 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3001 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3061 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3121 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3181 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3241 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3301 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     3361 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     3421 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     3481 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     3541 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     3601 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     3661 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     3721 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     3781 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     3841 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3901 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     3961 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4021 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4081 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4141 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4201 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4261 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     4321 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     4381 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     4441 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     4501 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     4561 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     4621 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     4681 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     4741 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     4801 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     4861 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     4921 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     4981 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5041 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5101 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5161 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     5221 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     5281 gtagaggatc g
//
