LOCUS       sequence-235140-        6560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6560)
  AUTHORS   Goldstone RM, Moreland NJ, Bashiri G, Baker EN, Shaun Lott J
  TITLE     A new Gateway vector and expression protocol for fast and efficient 
            recombinant protein expression in Mycobacterium smegmatis.
  JOURNAL   Protein Expr Purif. 2008 Jan . 57(1):81-7.
  PUBMED    17949993
REFERENCE   2  (bases 1 to 6560)
  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: "Protein 
            Expr Purif. 2008 Jan . 57(1):81-7."
COMMENT     Sequence Label: pDESTsmg
FEATURES             Location/Qualifiers
     source          1..6560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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)."
     RBS             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    187..311
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(336..341,342..359,360..366)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             420..1079
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(486..505)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1421..1726
                     /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     1605..1624
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1767..1891)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1941..1958
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2011..2029)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2025..2072
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2109..2537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2196..2215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2406..2427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      complement(4640..5228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4720..4739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atataccatg tcgcatcacc atcaccatca cctcgaatca
      181 ccatcaacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
      241 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
      301 cagtcactat ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt
      361 ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa
      421 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
      481 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
      541 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
      601 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
      661 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
      721 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
      781 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
      841 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
      901 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
      961 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     1021 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa
     1081 gatctggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt
     1141 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat
     1201 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat
     1261 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt
     1321 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa
     1381 atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac
     1441 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1501 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     1561 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     1621 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     1681 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct
     1741 tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt
     1801 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     1861 tttctcgttc agctttcttg tacaaagtgg ttgatgatcc gaattcgagc tccgtcgaca
     1921 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1981 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2041 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2101 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2161 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2221 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2281 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2341 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2401 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2461 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2521 aaaatttaac gcgaatttta acaaaatatc tagatttaaa gatctggtac cgcggccgcg
     2581 ccacggatgc caccacaggc actacaacgg agttcgccac gtacatcacc acaaccaccg
     2641 attctggcgg tgagctcccc gatattcagc ggaaatggct tggtatcgac caagattcgt
     2701 agaaccccgt ctcgtctggc tggtattcaa aacgggcgca acgaaacacg caacgagaca
     2761 ggcatggccc aaaccagaaa actagcgtct accaggactt ttacctgtcc gacccgttgc
     2821 aacggaaccc cccacggaac ccccgcgaca cccgctcccc aattgcgtta gaacagcggt
     2881 ggattgtcgg cttcgttgtg ggccttttga gccgcttcct gttctgccgc acgctctttc
     2941 ctcgcccgat agccgagtcg cttaacggtg tccagatgca gcccgaaatg tttggccgtt
     3001 tgcggccaag agtggccctc gtcgtcgtga taggcgcgga tgcgttcgcg gcgtgcagcc
     3061 tgctcggcga gccactcgct gcgttcctgc gccacgagcc ggacgacgtg gcgttcggat
     3121 agtccggtga ttcgagcgcc ttcggcggcg gtcacgcgcc gctttttgcg gacagtcggc
     3181 tgccggttgt agccgtcgct gtagccgtcg ctcatagcaa tgcctccatg catggctgac
     3241 gcggactttg cgcgccgcgc aactgtgctc gccgccgtgc gcgctgctgc gcccttccgc
     3301 gagatggccg actggcgcgc actgagtgtg gcctcgtaga ccacgatccc gtccgcccaa
     3361 atgcgcgact tggttgtgat ccaacgccaa atgctgttgg cgatggcgcg gacctcgctg
     3421 tccggtagcg gtccgggaca cacgtcgttg cacggaaatt cggcgtttcg cgcgtggcac
     3481 tcggcataga tcgcgcggcc gagtccgtcc acgttccggg tcggcaggta gatccgcatg
     3541 agggcgggac gataggccca caacctgacg gaatcgaaca gtgcgcaatt ccgccctagc
     3601 ggcgtcggag ccgctttgta cgtggtctgc tgacgccagc gcggcggtgg catgttcgcg
     3661 ccgagctcgg cctcgatgtg gctgagtgtg tagagatctg agtggagcca ttccgtttcc
     3721 caggcgatgt ggccggggtt tttggtcatg aggcctgagt aactgcggtc gccatcgacg
     3781 gcgcgccgaa ggccttcggc gcacgccgcc atgtatgcga gcggcttacg ccgcgcgtat
     3841 tcggtgcgtg gaacaggggc gttgagtgcc cacactgcgt gtgcgtggcc gttggcgcga
     3901 ttgcccacga tcgcgttggg cagcggatgg gacccccggg cgctgagcgc tcggagcgct
     3961 gcgtctggat ggtctacgtc cacgaccagc aggtttgcca gcgctgttgg gttcgcctcg
     4021 atgtaccggc ggcctagggc cgacgcgcgg ctttggcggt agatcccctc gagcagatcg
     4081 tcgcttgcca gcggccagta cggcagccag agctgctcaa attcgtcggc gacgtggctc
     4141 acgcttggta gtagaccacg attaatcacc ggtgtatggt ccgacacgag ctccaagtca
     4201 gatatttcgc tgaggggcca ccccacaact gcacactccc ccgctctccc gtcgagccct
     4261 ggtggtggaa caccagcgac agccgagcac ccccaaccac ctgtaccaac caggaggaac
     4321 acatgcgtcg tttcgaggac gtttccgggc cgctgagagc cgctgtggcg gccgtacacg
     4381 ccgccttaga cccgttagac cccctgccgc ctgaatgcgc gggtacgagc cacacagcgc
     4441 ccgaacttac ggagctggtg ggctcacgcg tgcggccgca cgcgttgcgc tcggtcgttc
     4501 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4561 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4801 cctttctccc ctcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5041 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcatgga actagagcat
     5281 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5341 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5401 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgacgtggc
     5461 cgaccagccc gtcatcgtca acgcctgatc cgcggtgcgg acaggccgtg tcgcgaccgg
     5521 ccgtgcggaa ttaagccggc ccgtaccctg tgaatagagg tccgctgtga cacaagaatc
     5581 cctgttactt ctcgaccgta ttgattcgga tgattcctac gcgagcctgc ggaacgacca
     5641 ggaattctgg gagccgctgg cccgccgagc cctggaggag ctcgggctgc cggtgccgcc
     5701 ggtgctgcgg gtgcccggcg agagcaccaa ccccgtactg gtcggcgagc ccggcccggt
     5761 gatcaagctg ttcggcgagc actggtgcgg tccggagagc ctcgcgtcgg agtcggaggc
     5821 gtacgcggtc ctggcggacg ccccggtgcc ggtgccccgc ctcctcggcc gcggcgagct
     5881 gcggcccggc accggagcct ggccgtggcc ctacctggtg atgagccgga tgaccggcac
     5941 cacctggcgg tccgcgatgg acggcacgac cgaccggaac gcgctgctcg ccctggcccg
     6001 cgaactcggc cgggtgctcg gccggctgca cagggtgccg ctgaccggga acaccgtgct
     6061 caccccccat tccgaggtct tcccggaact gctgcgggaa cgccgcgcgg cgaccgtcga
     6121 ggaccaccgc gggtggggct acctctcgcc ccggctgctg gaccgcctgg aggactggct
     6181 gccggacgtg gacacgctgc tggccggccg cgaaccccgg ttcgtccacg gcgacctgca
     6241 cgggaccaac atcttcgtgg acctggccgc gaccgaggtc accgggatcg tcgacttcac
     6301 cgacgtctat gcgggagact cccgctacag cctggtgcaa ctgcatctca acgccttccg
     6361 gggcgaccgc gagatcctgg ccgcgctgct cgacggggcg cagtggaagc ggaccgagga
     6421 cttcgcccgc gaactgctcg ccttcacctt cctgcacgac ttcgaggtgt tcgaggagac
     6481 cccgctggat ctctccggct tcaccgatcc ggaggaactg gcgcagttcc tctgggggcc
     6541 gccggacacc gcccccggcg
//
