LOCUS       sequence-211292-        8256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Analogous to pMINTNH3 but with a C-terminal 3C cleavage site and a 
            GFP-His10 tag fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8256)
  AUTHORS   Arnold FM, Hohl M, Remm S, Koliwer-Brandl H, Adenau S, Chusri S, 
            Sander P, Hilbi H, Seeger MA
  TITLE     A uniform cloning platform for mycobacterial genetics and protein 
            production.
  JOURNAL   Sci Rep. 2018 Jun 22;8(1):9539. doi: 10.1038/s41598-018-27687-5.
  PUBMED    29934571
REFERENCE   2  (bases 1 to 8256)
  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: "Sci Rep."; 
            date: "2018-06-22"; volume: "8(1):9539. doi"; pages: " 
            10.1038/s41598-018-27687-5"
COMMENT     Sequence Label: pMINTC3GH (Kan)
FEATURES             Location/Qualifiers
     source          1..8256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    161..185
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(574..598)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(677..982)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(779..798)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1324..1983)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1898..1917
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2037..2043,2044..2061,2062..2067))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2092..2216)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2235..2255)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2259..2279)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2306..2329
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2336..3046
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2363..2391)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2986..3006
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3053..3082
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     rep_origin      complement(3352..3940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3432..3451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4271..5086)
                     /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
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4994..5013
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5078..5097
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(7033..7079)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7335..7357
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7354..7370
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7354..7370
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7390..7410
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     7463..7479
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7464..7480
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaagtgattc ctcctgatgg taccggatcc gtggcaggag ggtcgcctgc gtcggcaact
       61 cgcccaggct acacgagagc cctcgactgc gaaaatgccg accaggcacc accgcactcc
      121 aggcttaacc cggtgcgacg atccaatatt acgagatctg acaagtttgt acaaaaaagc
      181 aggctaccag gcctagatct ggggacccta gaggtcccct tttttatttt aaaaattttt
      241 tcacgtgcca gccgattgga ctagaaatat tggatcgtcg gcaccgtcac ggccgtggga
      301 ggcggcacga tccgcgacgt gatgatcggc cgcatcccca cggtgctgcg cagtgagctc
      361 tacgccatcc cggcgttgat ctgtgcgttc gcacgcacag gcccggtgtg agaagggtct
      421 ctgacgagcg ggagaactcc ctatcagtga tagagtttgt cctccctatc agtgatagat
      481 aggctctggg agtacccgtg tgtacgacca gcacggcata catcatttcg acgccgagag
      541 attcgccgcc cgaaatgagc acgatccgca tgcacccagc tttcttgtac aaagtgggct
      601 cgaggcatgc gaaggagata tacatatgag ttgaagagcg acctgcagac tggctgtgta
      661 taagggagcc tgacatttat attccccaga acatcaggtt aatggcgttt ttgatgtcat
      721 tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc acactggcca
      781 tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg taaagttcac
      841 gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc cgtcgcccgg
      901 gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc tctcttttat
      961 aggtgtaaac cttaaactgc atttcaccag cccctgttct cgtcagcaaa agagccgttc
     1021 atttcaataa accgggcgac ctcagccatc ccttcctgat tttccgcttt ccagcgttcg
     1081 gcacgcagac gacgggcttc attctgcatg gttgtgctta ccagaccgga gatattgaca
     1141 tcatatatgc cttgagcaac tgatagctgt cgctgtcaac tgtcactgta atacgctgct
     1201 tcatagcata cctctttttg acatacttcg ggtatacata tcagtatata ttcttatacc
     1261 gcaaaaatca gcgcgcaaat acgcatactg ttatctggct tttagtaagc cggatccacg
     1321 cgtttacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc
     1381 gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt
     1441 gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt
     1501 ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat
     1561 attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg
     1621 cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa
     1681 cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag
     1741 ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc gggcaagaat
     1801 gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt
     1861 aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa
     1921 atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc
     1981 cattttagct tccttagctc ctgaaaatct cgacggatcc taactcaaaa tccacacatt
     2041 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgcggccg ccatagtgac
     2101 tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat
     2161 atattgatat ttatatcatt ttacgtttct cgttcagctt ttttgtacaa acttgtgata
     2221 tcgaattcgc tagctacctt tctcttcttt tttggatcta cctttctctt cttttttggg
     2281 gatccactag ttcagctctt ctgcattaga agttttgttt caaggtccac aattcagcaa
     2341 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     2401 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gctacatacg gaaaactcac
     2461 ccttaaattt atttgcacta ctggaaaact acctgttcct tggccaacac ttgtcactac
     2521 tctgacctat ggtgttcaat gcttttcccg ttatccggat cacatgaaac ggcatgactt
     2581 tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga
     2641 cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat
     2701 cgagttaaag ggtattgatt ttaaagaaga tggaaacatt ctcggacaca aactagagta
     2761 caactataac tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc
     2821 taacttcaaa attcgccaca acattgaaga tggttccgtt caactagcag accattatca
     2881 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtcgac
     2941 acaatctgcc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc ttcttgagtt
     3001 tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaaacta gtcatcatca
     3061 tcaccatcat catcaccatc attaattata catagttgct taagctgcag gggaccctag
     3121 aggtcccctt ttttatttta aaaatttttt ataaccggta agaagtccaa cacgccgctc
     3181 ctccccgatg ttgcgtagcc gcttcaagct taagcaacgg ccccagtgag tagcgacttt
     3241 ggatccggta cccgcaacgg tcaaccagcg tctacgcgtt tcactggccg acatgtgagc
     3301 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3361 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3421 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3481 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccctcgggaa gcgtggcgct
     3541 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3601 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3661 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3721 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3781 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3841 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3901 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3961 tacggggtct gacgctcatg gaactagtga ggtctgcctc gtgaagaagg tgttgctgac
     4021 tcataccagg cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttggtg
     4081 agagctttgt tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg
     4141 tctgcgttgt cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt
     4201 caacaaagcc gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa
     4261 ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     4321 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     4381 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     4441 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     4501 gagtgacgac tgaatccggt gagaatggca aaagattatg catttctttc cagacttgtt
     4561 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     4621 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     4681 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     4741 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     4801 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     4861 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     4921 gtttcagaaa caactctggc gcatggggct tcccatacaa gcgatagatt gtcgcacctg
     4981 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     5041 ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     5101 tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa
     5161 tgtaacatca gagattttga gacacaacgt ggctttcccc ccccccgacg tcaggtggct
     5221 agctggcggg aacggcaatg ctcctggcgg cggcggggct ggtgcccagg tctccgcaca
     5281 gagcggcggt gctggcgctc gcggccaggc gtggttcttc gcgtactgac aagaaacccc
     5341 cctctttagg actcagtgtc cttgggaggg gggctttttg cgtttcagga ggtcttggcc
     5401 agcttggaca tcgcctcagc gatagcctcg tcgcgggcct cagacgccat ctggtacttc
     5461 atcgccatcc taggagtcgt gtgaccgaga cgggccatca gctccttggt cgtcgcacct
     5521 gcctgagcgg cgaacgtagc gccgacagcg cggaggtcgt ggatgcggag ttccggccga
     5581 ccgatcttgg cgtagccacg cttcagcgac ttggtgaacg cggacttcga cagccggttg
     5641 ccctgcgtcg tggtcaccag gaatgcctcg gggcccttgt tcatcttcgt acggtccttc
     5701 atgtgcgctc ggatcatctc cgcgacgtga ggcggaaccg tcacaggacg cttcgaccgg
     5761 acggtcttgg cgttgccaac gacgatcttg ttccccacgc gggaagcgcc acggcgcacc
     5821 cggagcttca tcgtcatgcc gtcgtccacg atgtccttgc ggcgaagctc gatcagctct
     5881 ccgaaccgga ggctcgtcca cgccaggatg tatgccgcga tccggtagtg cacgaagatc
     5941 tcagcggcga cgatgtccag ctcctcaggc gtcagcgcct ctacgtcgcg ctcatcggct
     6001 gccttctgct cgatccggca cgggttctct gcgatcagct tgtcctcgac cgctgtgttc
     6061 atcaccgccc ggaggacgtt gtaggcatgc cggcgggcag tcgggtgctt cctacccatc
     6121 ccggcccacc acgcacgcac cagagctggc gtcatctctg tgaccgccac ttcacctagc
     6181 accgggtaga tgcggcgctc cgcgtgcccg ctgtacagat ccctggtgcc gtctgcgagg
     6241 tcgcgctcca cgagccactt ccgggtgtac tcctccagcg tgatggcgct ggcggctgcc
     6301 ttcttcgccc ggtcctgtgg aggggtccag gtctccatct cgatgagccg cttctcgccc
     6361 gcgagccagg cttcggcgtc catcttgttg tcgtaggtct gcagcgcgta gtacctcaca
     6421 ccgtcctgcg ggttgacgta tgaggcttgg atcctcccgc tgcgctgagt cttcagcgat
     6481 ccccatccgc gacgtgccaa ctaggtctcc tctcgtcgtg aacaaggcta ccgggttgca
     6541 actcctgtgc aactctcagg cttcaacgcg cttctacgac ctgcaatttc tttccactta
     6601 gaggatgcag ccgagagggg gtaaaaacct atcttgaccg gcccatatat gtggtcggca
     6661 gacacccatt cttccaaact agctacgcgg gttcgattcc cgtcgcccgc tccgctggtc
     6721 agagggtgtt ttcgccctct ggccattttt ctttccaggg gtctgcaact cttgtgcgac
     6781 tcttctgacc tgggcatacg cggttgcaac gcatccctga tctggctact ttcgatgctg
     6841 acaaacgaat agagcccccc gcctgcgcga acagacgagg ggcattcaca ccagattgga
     6901 gctggtgcag tgaagagaat agaccgggac aaggttgcac cgggagttgc agcggtcgga
     6961 accctcgccg tcggcgggct ggcgttcgcc ctgtcgttca cggctctagc tgatcaccgc
     7021 ggccatgatg gcataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
     7081 tgcctggcag tcgatcgtac gctagttaac tacgtcgaca tcgatttaat taatatgcat
     7141 caattgattt aaatactagt gttaactatt taattgggga ccctagaggt cccctttttt
     7201 attttaaaaa ttttttcaag gggacctcta gggtccccag ctggctagcg agtcatgagg
     7261 tcaccatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7321 agtgagctga taccagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     7381 ccaagctatc aactttgtat agaaaagttg catgaccaac ttcgataacg ttctcggctc
     7441 gatgatcccc gggggccccc cctcgaggtc gacggtatcg atttaattaa tatgcatcaa
     7501 ttgatttaaa tactagtgtt aactatttaa ttggggaccc tagaggtccc cttttttatt
     7561 ttaaaaattt tttcaagggg acctctaggg tccccagctg gctagcgagt catgaggtca
     7621 ccatggtcat taggagccgc tctcgcactt cagctgcttc tccaagccgc agatgatcag
     7681 ctccaggccg aacaagaagg ccggctcggc gccctggtgg tcgaacagct cgatggcctg
     7741 gcgcagcaac ggcggcatgc tgtcggtggt cggggtctcg cgctcctcct tggcgacctg
     7801 gtgctcctgg tcctccaaca cgcagcccag ggtgaagtgg cccacggccg acagggcgta
     7861 caacgcgttc tccagcgaga agccctgctg gcacaggaag gccaactggt tctccagggt
     7921 ctcgtactgc ttctcggtcg gccgggtgcc caggtgcacc ttggcgccgt cgcggtggct
     7981 cagcaaggcg cagcggaagc tcttggcgtt gttgcgcagg aagtcctgcc agctctcgcc
     8041 ctccagcggg cagaagtggg tgtggtggcg gtccaacatc tcgatggcca gggcgtcgag
     8101 cagggcccgc ttgttcttga cgtgccagta cagggtcggc tgctcgacgc ccagcttctg
     8161 ggccagcttg cgggtggtca ggccctcgat gccgacctcg ttcagcagct ccagggcgga
     8221 gttgatgacc ttggacttgt ccaggcggga cattgc
//
