LOCUS       sequence-417380-        3949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for cloning of FatI digested metagenomic DNA into
            a compatible NcoI site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3949)
  AUTHORS   Rich MH, Sharrock AV, Mulligan TS, Matthews F, Brown AS, Lee-Harwood
            HR, Williams EM, Copp JN, Little RF, Francis JJ, Horvat CN, 
            Stevenson LJ, Owen JG, Saxena MT, Mumm JS, Ackerley DF
  TITLE     A metagenomic library cloning strategy that promotes high-level 
            expression of captured genes to enable efficient functional 
            screening.
  JOURNAL   bioRxiv. 2023 Mar 25:2023.03.24.534183. doi: 
            10.1101/2023.03.24.534183. Preprint.
  PUBMED    36993673
REFERENCE   2  (bases 1 to 3949)
  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. 
            2023 Mar 25:2023.03.24.534183. doi: 10.1101/2023.03.24.534183. 
            Preprint."
COMMENT     Sequence Label: pUCXMG
FEATURES             Location/Qualifiers
     source          1..3949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..33
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     primer_bind     complement(100..117)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      270..356
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      448..475
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(550..569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     669..691
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(729..747)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        815..919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             920..1453
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     rep_origin      1624..2212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2113..2132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2366..2383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2403..3485)
                     /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     3447..3466
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3486..3563)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        join(3793..3798,3799..3814,3815..3821)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3825..3849
                     /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             3864..3886
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3905..3922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3932..3949
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga
       61 caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
      121 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
      181 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
      241 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
      301 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
      361 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
      421 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
      481 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gactagtgtg
      541 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
      601 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      661 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      721 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
      781 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
      841 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      901 atattgaaaa aggaagagta tgttacgcag cagcaacgat gttacgcagc agggcagtcg
      961 ccctaaaaca aagttaggtg gctcaagtat gggcatcatt cgcacatgta ggctcggccc
     1021 tgaccaagtc aaatccatgc gggctgctct tgatcttttc ggtcgtgagt tcggagacgt
     1081 agccacctac tcccaacatc agccggactc cgattacctc gggaacttgc tccgtagtaa
     1141 gacattcatc gcgcttgctg ccttcgacca agaagcggtt gttggcgctc tcgcggctta
     1201 cgttctgccc aagtttgagc agccgcgtag tgagatctat atctatgatc tcgcagtctc
     1261 cggcgagcac cggaggcagg gcattgccac cgcgctcatc aatctcctca agcatgaggc
     1321 caacgcgctt ggtgcttatg tgatctacgt gcaagcagat tacggtgacg atcccgcagt
     1381 ggctctctat acaaagttgg gcatacggga agaagtgatg cactttgata tcgacccaag
     1441 taccgccacc taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1501 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1561 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1621 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1681 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1741 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     1801 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1861 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1921 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1981 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2041 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2101 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2161 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2221 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2281 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2341 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcaccg
     2401 gttcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2461 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     2521 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     2581 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     2641 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     2701 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     2761 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     2821 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     2881 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     2941 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     3001 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     3061 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     3121 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     3181 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     3241 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     3301 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     3361 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     3421 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     3481 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     3541 gttttgcacc attcgatggt gtcaacgtaa atgccgcttc gccttcgcgc gcgaattgca
     3601 agctgatccg ggcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     3661 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     3721 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     3781 tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat
     3841 aacaattccc ctctagaaat aactttgttt aactttaaga aggagatata cgatgggcag
     3901 cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagc
//
