LOCUS       sequence-269882-        8599 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 8599)
  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 8599)
  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 (Apr)
FEATURES             Location/Qualifiers
     source          1..8599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(254..278)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(357..662)
                     /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(459..478)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1004..1663)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1578..1597
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1717..1723,1724..1741,1742..1747))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1772..1896)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1915..1935)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1939..1959)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1986..2009
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2016..2726
                     /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(2043..2071)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2666..2686
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2733..2762
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     rep_origin      complement(3032..3620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3112..3131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             4108..4908
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     primer_bind     5101..5120
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(7056..7102)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7358..7380
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7377..7393
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7377..7393
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7413..7433
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     7486..7502
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7487..7503
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8440..8464
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gatgatcggc cgcatcccca cggtgctgcg cagtgagctc tacgccatcc cggcgttgat
       61 ctgtgcgttc gcacgcacag gcccggtgtg agaagggtct ctgacgagcg ggagaactcc
      121 ctatcagtga tagagtttgt cctccctatc agtgatagat aggctctggg agtacccgtg
      181 tgtacgacca gcacggcata catcatttcg acgccgagag attcgccgcc cgaaatgagc
      241 acgatccgca tgcacccagc tttcttgtac aaagtgggct cgaggcatgc gaaggagata
      301 tacatatgag ttgaagagcg acctgcagac tggctgtgta taagggagcc tgacatttat
      361 attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca
      421 gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc
      481 cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc
      541 agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc
      601 tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc
      661 atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac
      721 ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc
      781 attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac
      841 tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg
      901 acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat
      961 acgcatactg ttatctggct tttagtaagc cggatccacg cgtttacgcc ccgccctgcc
     1021 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     1081 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     1141 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     1201 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     1261 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact
     1321 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     1381 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     1441 tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     1501 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     1561 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     1621 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc
     1681 ctgaaaatct cgacggatcc taactcaaaa tccacacatt atacgagccg gaagcataaa
     1741 gtgtaaagcc tggggtgcct aatgcggccg ccatagtgac tggatatgtt gtgttttaca
     1801 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     1861 ttacgtttct cgttcagctt ttttgtacaa acttgtgata tcgaattcgc tagctacctt
     1921 tctcttcttt tttggatcta cctttctctt cttttttggg gatccactag ttcagctctt
     1981 ctgcattaga agttttgttt caaggtccac aattcagcaa aggagaagaa cttttcactg
     2041 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     2101 gtggagaggg tgaaggtgat gctacatacg gaaaactcac ccttaaattt atttgcacta
     2161 ctggaaaact acctgttcct tggccaacac ttgtcactac tctgacctat ggtgttcaat
     2221 gcttttcccg ttatccggat cacatgaaac ggcatgactt tttcaagagt gccatgcccg
     2281 aaggttatgt acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtg
     2341 ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat cgagttaaag ggtattgatt
     2401 ttaaagaaga tggaaacatt ctcggacaca aactagagta caactataac tcacacaatg
     2461 tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccaca
     2521 acattgaaga tggttccgtt caactagcag accattatca acaaaatact ccaattggcg
     2581 atggccctgt ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaag
     2641 atcccaacga aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggatta
     2701 cacatggcat ggatgagctc tacaaaacta gtcatcatca tcaccatcat catcaccatc
     2761 attaattata catagttgct taagctgcag gggaccctag aggtcccctt ttttatttta
     2821 aaaatttttt ataaccggta agaagtccaa cacgccgctc ctccccgatg ttgcgtagcc
     2881 gcttcaagct taagcaacgg ccccagtgag tagcgacttt ggatccggta cccgcaacgg
     2941 tcaaccagcg tctacgcgtt tcactggccg acatgtgagc aaaaggccag caaaaggcca
     3001 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3061 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3121 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3181 gatacctgtc cgcctttctc ccctcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3241 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3301 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3361 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3421 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     3481 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3541 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3601 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcatg
     3661 gaactagtga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg cctgaatcgc
     3721 cccatcatcc agccagaaag tgagggagcc acggttggtg agagctttgt tgtaggtgga
     3781 ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt cgggaagatg
     3841 cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt
     3901 caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga ttagaaaaac
     3961 tcatcgagca tcaaatgaaa ctgcatctag aaggatcttc acctagatcc ttttggttca
     4021 tgtgcagctc catcagcaaa aggggatgat aagtttatca ccaccgacta tttgcaacag
     4081 tgccgttgat cgtgctatga tcgactgatg tcatcagcgg tggagtgcaa tgtcgtgcaa
     4141 tacgaatggc gaaaagccga gctcatcggt cagcttctca accttggggt tacccccggc
     4201 ggtgtgctgc tggtccacag ctccttccgt agcgtccggc ccctcgaaga tgggccactt
     4261 ggactgatcg aggccctgcg tgctgcgctg ggtccgggag ggacgctcgt catgccctcg
     4321 tggtcaggtc tggacgacga gccgttcgat cctgccacgt cgcccgttac accggacctt
     4381 ggagttgtct ctgacacatt ctggcgcctg ccaaatgtaa agcgcagcgc ccatccattt
     4441 gcctttgcgg cagcggggcc acaggcagag cagatcatct ctgatccatt gcccctgcca
     4501 cctcactcgc ctgcaagccc ggtcgcccgt gtccatgaac tcgatgggca ggtacttctc
     4561 ctcggcgtgg gacacgatgc caacacgacg ctgcatcttg ccgagttgat ggcaaaggtt
     4621 ccctatgggg tgccgagaca ctgcaccatt cttcaggatg gcaagttggt acgcgtcgat
     4681 tatctcgaga atgaccactg ctgtgagcgc tttgccttgg cggacaggtg gctcaaggaa
     4741 aagagccttc agaaggaagg tccagtcggt catgcctttg ctcggttgat ccgctcccgc
     4801 gacattgtgg cgacagccct gggtcaactg ggccgagatc cgttgatctt cctgcatccg
     4861 ccagaggcgg gatgcgaaga atgcgatgcc gctcgccagt cgattggctg agctcatgag
     4921 cggagaacga gatgacgttg gaggggcaag gtcgcgctga ttgctggggc aacacgtgga
     4981 gcggatcggg gattgtcttt cttcagctcg ctgatgatat gctgacgctc aatgccgttt
     5041 ggcctccgac taacgaaaat cccgcatttg gacggctgat ccatggtact cccgttgaat
     5101 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     5161 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc
     5221 cccccccccg acgtcaggtg gctagctggc gggaacggca atgctcctgg cggcggcggg
     5281 gctggtgccc aggtctccgc acagagcggc ggtgctggcg ctcgcggcca ggcgtggttc
     5341 ttcgcgtact gacaagaaac ccccctcttt aggactcagt gtccttggga ggggggcttt
     5401 ttgcgtttca ggaggtcttg gccagcttgg acatcgcctc agcgatagcc tcgtcgcggg
     5461 cctcagacgc catctggtac ttcatcgcca tcctaggagt cgtgtgaccg agacgggcca
     5521 tcagctcctt ggtcgtcgca cctgcctgag cggcgaacgt agcgccgaca gcgcggaggt
     5581 cgtggatgcg gagttccggc cgaccgatct tggcgtagcc acgcttcagc gacttggtga
     5641 acgcggactt cgacagccgg ttgccctgcg tcgtggtcac caggaatgcc tcggggccct
     5701 tgttcatctt cgtacggtcc ttcatgtgcg ctcggatcat ctccgcgacg tgaggcggaa
     5761 ccgtcacagg acgcttcgac cggacggtct tggcgttgcc aacgacgatc ttgttcccca
     5821 cgcgggaagc gccacggcgc acccggagct tcatcgtcat gccgtcgtcc acgatgtcct
     5881 tgcggcgaag ctcgatcagc tctccgaacc ggaggctcgt ccacgccagg atgtatgccg
     5941 cgatccggta gtgcacgaag atctcagcgg cgacgatgtc cagctcctca ggcgtcagcg
     6001 cctctacgtc gcgctcatcg gctgccttct gctcgatccg gcacgggttc tctgcgatca
     6061 gcttgtcctc gaccgctgtg ttcatcaccg cccggaggac gttgtaggca tgccggcggg
     6121 cagtcgggtg cttcctaccc atcccggccc accacgcacg caccagagct ggcgtcatct
     6181 ctgtgaccgc cacttcacct agcaccgggt agatgcggcg ctccgcgtgc ccgctgtaca
     6241 gatccctggt gccgtctgcg aggtcgcgct ccacgagcca cttccgggtg tactcctcca
     6301 gcgtgatggc gctggcggct gccttcttcg cccggtcctg tggaggggtc caggtctcca
     6361 tctcgatgag ccgcttctcg cccgcgagcc aggcttcggc gtccatcttg ttgtcgtagg
     6421 tctgcagcgc gtagtacctc acaccgtcct gcgggttgac gtatgaggct tggatcctcc
     6481 cgctgcgctg agtcttcagc gatccccatc cgcgacgtgc caactaggtc tcctctcgtc
     6541 gtgaacaagg ctaccgggtt gcaactcctg tgcaactctc aggcttcaac gcgcttctac
     6601 gacctgcaat ttctttccac ttagaggatg cagccgagag ggggtaaaaa cctatcttga
     6661 ccggcccata tatgtggtcg gcagacaccc attcttccaa actagctacg cgggttcgat
     6721 tcccgtcgcc cgctccgctg gtcagagggt gttttcgccc tctggccatt tttctttcca
     6781 ggggtctgca actcttgtgc gactcttctg acctgggcat acgcggttgc aacgcatccc
     6841 tgatctggct actttcgatg ctgacaaacg aatagagccc cccgcctgcg cgaacagacg
     6901 aggggcattc acaccagatt ggagctggtg cagtgaagag aatagaccgg gacaaggttg
     6961 caccgggagt tgcagcggtc ggaaccctcg ccgtcggcgg gctggcgttc gccctgtcgt
     7021 tcacggctct agctgatcac cgcggccatg atggcataaa acgaaaggcc cagtctttcg
     7081 actgagcctt tcgttttatt tgatgcctgg cagtcgatcg tacgctagtt aactacgtcg
     7141 acatcgattt aattaatatg catcaattga tttaaatact agtgttaact atttaattgg
     7201 ggaccctaga ggtccccttt tttattttaa aaattttttc aaggggacct ctagggtccc
     7261 cagctggcta gcgagtcatg aggtcaccat gttctttcct gcgttatccc ctgattctgt
     7321 ggataaccgt attaccgcct ttgagtgagc tgataccagc ggataacaat ttcacacagg
     7381 aaacagctat gaccatgatt acgccaagct atcaactttg tatagaaaag ttgcatgacc
     7441 aacttcgata acgttctcgg ctcgatgatc cccgggggcc ccccctcgag gtcgacggta
     7501 tcgatttaat taatatgcat caattgattt aaatactagt gttaactatt taattgggga
     7561 ccctagaggt cccctttttt attttaaaaa ttttttcaag gggacctcta gggtccccag
     7621 ctggctagcg agtcatgagg tcaccatggt cattaggagc cgctctcgca cttcagctgc
     7681 ttctccaagc cgcagatgat cagctccagg ccgaacaaga aggccggctc ggcgccctgg
     7741 tggtcgaaca gctcgatggc ctggcgcagc aacggcggca tgctgtcggt ggtcggggtc
     7801 tcgcgctcct ccttggcgac ctggtgctcc tggtcctcca acacgcagcc cagggtgaag
     7861 tggcccacgg ccgacagggc gtacaacgcg ttctccagcg agaagccctg ctggcacagg
     7921 aaggccaact ggttctccag ggtctcgtac tgcttctcgg tcggccgggt gcccaggtgc
     7981 accttggcgc cgtcgcggtg gctcagcaag gcgcagcgga agctcttggc gttgttgcgc
     8041 aggaagtcct gccagctctc gccctccagc gggcagaagt gggtgtggtg gcggtccaac
     8101 atctcgatgg ccagggcgtc gagcagggcc cgcttgttct tgacgtgcca gtacagggtc
     8161 ggctgctcga cgcccagctt ctgggccagc ttgcgggtgg tcaggccctc gatgccgacc
     8221 tcgttcagca gctccagggc ggagttgatg accttggact tgtccaggcg ggacattgcg
     8281 aagtgattcc tcctgatggt accggatccg tggcaggagg gtcgcctgcg tcggcaactc
     8341 gcccaggcta cacgagagcc ctcgactgcg aaaatgccga ccaggcacca ccgcactcca
     8401 ggcttaaccc ggtgcgacga tccaatatta cgagatctga caagtttgta caaaaaagca
     8461 ggctaccagg cctagatctg gggaccctag aggtcccctt ttttatttta aaaatttttt
     8521 cacgtgccag ccgattggac tagaaatatt ggatcgtcgg caccgtcacg gccgtgggag
     8581 gcggcacgat ccgcgacgt
//
