LOCUS       sequence-426303-        6022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with C terminal TEV cleavable sfGFP HisAsn6 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6022)
  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 6022)
  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-CT-sfGFP-HN-GG
FEATURES             Location/Qualifiers
     source          1..6022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    67..91
                     /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             168..188
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(201..203,204..914)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             915..950
                     /codon_start=1
                     /product="6x(His-Asn) affinity tag"
                     /label=6xHN
                     /translation="HNHNHNHNHNHN"
     primer_bind     complement(1004..1022)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1018..1065
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1102..1557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1189..1208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1399..1420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1649..2464)
                     /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(1649..2464)
                     /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     2372..2391
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2456..2475
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2586..3174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3075..3094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3328..3345
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3360..3499
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3425..3444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3585..3607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3601..3792)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4601..5683)
                     /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     5645..5664
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5684..5761)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5967..5986
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
       61 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaaccttaa
      121 gaaggagata tacacatgag agaccgcgga taattcgggt ctcactcgag aacctctact
      181 tccaatcggg tagcagcggt atgagcaaag gtgaagaact gtttaccggt gttgttccga
      241 ttctggttga actggatggt gatgttaatg gccacaaatt ttcagttcgt ggtgaaggcg
      301 aaggtgatgc aaccaatggt aaactgaccc tgaaatttat ctgtaccacc ggcaaactgc
      361 cggttccgtg gccgacactg gttaccacac tgacctatgg tgttcagtgt tttagccgtt
      421 atccggatca tatgaaacgc cacgattttt tcaaaagcgc aatgccggaa ggttatgttc
      481 aagaacgtac catctccttt aaagatgatg gcacctataa aacccgtgcc gaagttaaat
      541 ttgaaggtga taccctggtg aatcgcattg aactgaaagg catcgatttc aaagaagatg
      601 gtaatatcct gggccacaaa ctggaatata atttcaatag ccacaacgtg tatatcaccg
      661 cagacaaaca gaaaaatggc atcaaagcca actttaagat tcggcataat gttgaagatg
      721 gcagcgttca gctggcagat cattatcagc agaatacccc gattggtgat ggtccggttc
      781 tgctgccgga taatcattat ctgagcaccc agagcgttct gagcaaagat ccgaatgaaa
      841 aacgtgatca catggtgctg ctggaatttg ttaccgcagc agggattacc catggtatgg
      901 atgaactgta taaacacaac cataatcaca atcataacca caaccacaat taaggatccg
      961 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1021 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1081 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1141 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1201 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1261 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1321 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1381 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1441 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1501 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1561 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1621 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1681 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1741 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1801 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1861 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     1921 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     1981 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2041 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2101 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2161 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2221 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2281 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2341 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2401 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2461 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2521 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2581 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2641 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2701 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2761 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2821 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2881 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2941 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3001 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3061 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3121 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3181 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3241 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3301 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3361 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3421 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3481 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3541 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3601 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3661 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3721 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3781 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     3841 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     3901 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     3961 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4021 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4081 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4141 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4201 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4261 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4321 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4381 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4441 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4501 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4561 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4621 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4681 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4741 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     4801 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4861 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4921 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     4981 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5041 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5101 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5161 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5221 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5281 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5341 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5401 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5461 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5521 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5581 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5641 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5701 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     5761 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     5821 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5881 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5941 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6001 cctgtggcgc cggtgatgcc gg
//
