LOCUS       sequence-211576-        6523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Analogous to pFLAG_attP, but containing oriM instead of attP, thus 
            being a replicative vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  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 6523)
  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: pFLAG_OriM
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2008..2030
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2027..2043
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2027..2043
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2063..2083
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    complement(2510..2534)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2613..2918)
                     /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(2715..2734)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3260..3919)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3834..3853
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3973..3979,3980..3997,3998..4003))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4028..4152)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4171..4191)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4195..4215)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4242..4307
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4326..4343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      4418..4464
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      5837..6425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6326..6345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcaacgc
       61 gtgcggccgc acgcgtgagc ccaccagctc cgtaagttcg ggcgctgtgt ggctcgtacc
      121 cgcgcattca ggcggcaggg ggtctaacgg gtctaaggcg gcgtgtacgg ccgccacagc
      181 ggctctcagc ggcccggaaa cgtcctcgaa acgacgcatg tgttcctcct ggttggtaca
      241 ggtggttggg ggtgctcggc tgtcgctggt gttccaccac cagggctcga cgggagagcg
      301 ggggagtgtg cagttgtggg gtggcccctc agcgaaatat ctgacttgga gctcgtgtcg
      361 gaccatacac cggtgattaa tcgtggtcta ctaccaagcg tgagccacgt cgccgacgaa
      421 tttgagcagc tctggctgcc gtactggccg ctggcaagcg acgatctgct cgaggggatc
      481 taccgccaaa gccgcgcgtc ggccctaggc cgccggtaca tcgaggcgaa cccaacagcg
      541 ctggcaaacc tgctggtcgt ggacgtagac catccagacg cagcgctccg agcgctcagc
      601 gcccgggggt cccatccgct gcccaacgcg atcgtgggca atcgcgccaa cggccacgca
      661 cacgcagtgt gggcactcaa cgcccctgtt ccacgcaccg aatacgcgcg gcgtaagccg
      721 ctcgcataca tggcggcgtg cgccgaaggc cttcggcgcg ccgtcgatgg cgaccgcagt
      781 tactcaggcc tcatgaccaa aaaccccggc cacatcgcct gggaaacgga atggctccac
      841 tcagatctct acacactcag ccacatcgag gccgagctcg gcgcgaacat gccaccgccg
      901 cgctggcgtc agcagaccac gtacaaagcg gctccgacgc cgctagggcg gaattgcgca
      961 ctgttcgatt ccgtcaggtt gtgggcctat cgtcccgccc tcatgcggat ctacctgccg
     1021 acccggaacg tggacggact cggccgcgcg atctatgccg agtgccacgc gcgaaacgcc
     1081 gaatttccgt gcaacgacgt gtgtcccgga ccgctaccgg acagcgaggt ccgcgccatc
     1141 gccaacagca tttggcgttg gatcacaacc aagtcgcgca tttgggcgga cgggatcgtg
     1201 gtctacgagg ccacactcag tgcgcgccag tcggccatct cgcggaaggg cgcagcagcg
     1261 cgcacggcgg cgagcacagt tgcgcggcgc gcaaagtccg cgtcagccat gcatggaggc
     1321 attgctatga gcgacggcta cagcgacggc tacaaccggc agccgactgt ccgcaaaaag
     1381 cggcgcgtga ccgccgccga aggcgctcga atcaccggac tatccgaacg ccacgtcgtc
     1441 cggctcgtgg cgcaggaacg cagcgagtgg ctcgccgagc aggctgcacg ccgcgaacgc
     1501 atccgcgcct atcacgacga cgagggccac tcttggccgc aaacggccaa acatttcggg
     1561 ctgcatctgg acaccgttaa gcgactcggc tatcgggcga ggaaagagcg tgcggcagaa
     1621 caggaagcgg ctcaaaaggc ccacaacgaa gccgacaatc caccgctgtt ctaacgcaat
     1681 tggggagcgg gtgtcgcggg ggttccgtgg ggggttccgt tgcaacgggt cggacaggta
     1741 aaagtcctgg tagacgctag ttttctggtt tgggccatgc ctgtctcgtt gcgtgtttcg
     1801 ttgcgcccgt tttgaatacc agccagacga gacggggttc tacgaatctt ggtcgatacc
     1861 aagccatttc cgctgaatat cggggagctc accgccagaa tcggtggttg tggtgatgta
     1921 cgtggcgaac tccgttgtag tgcctgtggt ggcatccgtg gcgcggccgc ggtaccagat
     1981 ctttccgcct ttgagtgagc tgataccagc ggataacaat ttcacacagg aaacagctat
     2041 gaccatgatt acgccaagct atcaactttg tatagaaaag ttgcatgacc aacttcgata
     2101 acgttctcgg ctcgatgatc caccaggcct agatctgggg accctagagg tccccttttt
     2161 tattttaaaa attttttcac gtgccagccg attggactag aaatattgga tcgtcggcac
     2221 cgtcacggcc gtgggaggcg gcacgatccg cgacgtgatg atcggccgca tccccacggt
     2281 gctgcgcagt gagctctacg ccatcccggc gttgatctgt gcgttcgcac gcacaggccc
     2341 ggtgtgagaa gggtctctga cgagcgggag aactccctat cagtgataga gtttgtcctc
     2401 cctatcagtg atagataggc tctgggagta cccgtgtgta cgaccagcac ggcatacatc
     2461 atttcgacgc cgagagattc gccgcccgaa atgagcacga tccgcatgca cccagctttc
     2521 ttgtacaaag tgggctcgag gcatgcgaag gagatataca tatgagttga agagcgacct
     2581 gcagactggc tgtgtataag ggagcctgac atttatattc cccagaacat caggttaatg
     2641 gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc gataacggag
     2701 accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc gatatgcacc
     2761 accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc cagggggatc
     2821 accatccgtc gcccgggcgt gtcaataata tcactctgta catccacaaa cagacgataa
     2881 cggctctctc ttttataggt gtaaacctta aactgcattt caccagcccc tgttctcgtc
     2941 agcaaaagag ccgttcattt caataaaccg ggcgacctca gccatccctt cctgattttc
     3001 cgctttccag cgttcggcac gcagacgacg ggcttcattc tgcatggttg tgcttaccag
     3061 accggagata ttgacatcat atatgccttg agcaactgat agctgtcgct gtcaactgtc
     3121 actgtaatac gctgcttcat agcatacctc tttttgacat acttcgggta tacatatcag
     3181 tatatattct tataccgcaa aaatcagcgc gcaaatacgc atactgttat ctggctttta
     3241 gtaagccgga tccacgcgtt tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     3301 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     3361 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     3421 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     3481 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     3541 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     3601 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     3661 tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca
     3721 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     3781 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     3841 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     3901 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac ggatcctaac
     3961 tcaaaatcca cacattatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4021 cggccgccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg
     4081 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt cagctttttt
     4141 gtacaaactt gtgatatcga attcgctagc tacctttctc ttcttttttg gatctacctt
     4201 tctcttcttt tttggggatc cactagttct gctcttctgc agactacaaa gaccatgacg
     4261 gtgattataa agatcatgat atcgattaca aggatgacga tgacaagtag tgagcattcc
     4321 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4381 cactcaaagg cggagcgtac gatcgactgc caggcatcaa ataaaacgaa aggctcagtc
     4441 gaaagactgg gcctttcgtt ttatgccatc atggccgcgg tgatcagcta gagggggcgt
     4501 caggcgccgg gggcggtgtc cggcggcccc cagaggaact gcgccagttc ctccggatcg
     4561 gtgaagccgg agagatccag cggggtctcc tcgaacacct cgaagtcgtg caggaaggtg
     4621 aaggcgagca gttcgcgggc gaagtcctcg gtccgcttcc actgcgcccc gtcgagcagc
     4681 gcggccagga tctcgcggtc gccccggaag gcgttgagat gcagttgcac caggctgtag
     4741 cgggagtctc ccgcatagac gtcggtgaag tcgacgatcc cggtgacctc ggtcgcggcc
     4801 aggtccacga agatgttggt cccgtgcagg tcgccgtgga cgaaccgggg ttcgcggccg
     4861 gccagcagcg tgtccacgtc cggcagccag tcctccaggc ggtccagcag ccggggcgag
     4921 aggtagcccc acccgcggtg gtcctcgacg gtcgccgcgc ggcgttcccg cagcagttcc
     4981 gggaagacct cggaatgggg ggtgagcacg gtgttcccgg tcagcggcac cctgtgcagc
     5041 cggccgagca cccggccgag ttcgcgggcc agggcgagca gcgcgttccg gtcggtcgtg
     5101 ccgtccatcg cggaccgcca ggtggtgccg gtcatccggc tcatcaccag gtagggccac
     5161 ggccaggctc cggtgccggg ccgcagctcg ccgcggccga ggaggcgggg caccggcacc
     5221 ggggcgtccg ccaggaccgc gtacgcctcc gactccgacg cgaggctctc cggaccgcac
     5281 cagtgctcgc cgaacagctt gatcaccggg ccgggctcgc cgaccagtac ggggttggtg
     5341 ctctcgccgg gcacccgcag caccggcggc accggcagcc cgagctcctc cagggctcgg
     5401 cgggccagcg gctcccagaa ttcctggtcg ttccgcaggc tcgcgtagga atcatccgaa
     5461 tcaatacggt cgagaagtaa cagggattct tgtgtcacag cggacctcta ttcacagggt
     5521 acgggccggc ttaattccgc acggccggtc gcgacacggc ctgtccgcac cgcggatcag
     5581 gcgttgacga tgacgggctg gtcggccacg tcggggagtc aggcaactat ggatgaacga
     5641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5761 gtgaagatcc tttttgataa tctcatgctc tagttccatg agcgtcagac cccgtagaaa
     5821 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5881 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5941 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     6001 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6061 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6121 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6181 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6241 ccacgcttcc cgaggggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6301 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6361 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6421 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6481 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccg
//
