LOCUS       sequence-497169-        4952 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  P4Lacn.2 tagging casette for WTC848 with Nourseothricin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4952)
  TITLE     Stelling Lab Plasmids 2025
REFERENCE   2  (bases 1 to 4952)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: P2765
FEATURES             Location/Qualifiers
     source          1..4952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(336..1127,1128..1196))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     959..978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1197..1301)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1327..1782)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1464..1485)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1676..1695
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1909..1931
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1923..1940
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1924..1940
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1947..1966
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1947..1965
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            2025..3144
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2025..2368
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2369..2941
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2947..3144
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3775..3791
                     /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)."
     protein_bind    3804..3820
                     /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)."
     protein_bind    3896..3912
                     /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)."
     protein_bind    3917..3933
                     /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)."
     CDS             3954..4025
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     promoter        complement(4088..4106)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4127..4143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4127..4143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4140..4162)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4151..4167
                     /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)."
     promoter        complement(join(4175..4181,4182..4199,4200..4205))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4220..4241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4358..4375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4529..4952,1..165))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4609..4628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
       61 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      121 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      181 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      241 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      301 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      361 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      421 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
      481 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
      541 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      601 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      661 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      721 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      781 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      841 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      901 tcattctgag aatagtgtat gcggcgaccg agttgctctt gtccggcgtc aatacgggat
      961 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1021 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1081 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1141 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1201 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1261 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1321 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1381 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     1441 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1501 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1561 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1621 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     1681 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     1741 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     1801 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     1861 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     1921 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcg
     1981 atcctcgaga gcttgccttg tccccgccgg gtcacccggc cagcgacatg gaggcccaga
     2041 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
     2101 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
     2161 cggcgcgaag caaaaattac ggctcctcgc tgcggacctg cgagcaggga aacgctcccc
     2221 tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg
     2281 atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt
     2341 ctcacatcac atccgaacat aaacaaccat gggtaccact cttgacgaca cggcttaccg
     2401 gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg ggtccttcac
     2461 caccgacacc gtcttccgcg tcaccgccac cggggacggc ttcaccctgc gggaggtgcc
     2521 ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg aatcggacga
     2581 cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg acggcgacct
     2641 ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg tcgaggacat
     2701 cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg ggctcgcgac
     2761 ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca acgtcaacgc
     2821 accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg acaccgccct
     2881 gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc cctgccccta
     2941 atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt
     3001 tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc
     3061 gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt
     3121 atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgttt
     3181 aaacgaacta gtgcggccgc cagttcgagt ttatcattat caatactgcc atttcaaaga
     3241 atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagccta
     3301 tataacatcg taggtgtctg ggtgaacagt ttattcctgg catccactaa ttttaattct
     3361 gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag
     3421 gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa
     3481 gctggcatcc actaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat
     3541 cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa
     3601 aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca
     3661 attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc
     3721 tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttaattgtga
     3781 gcggataaca aagtataaat agattgtgag cggataacaa ttctgtaaat ctatttctta
     3841 aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac accaattgtg
     3901 agcggataac aattaattgt gagcggataa caattacaca cataaacaaa atggattata
     3961 aagatgacga tgacaaggat tataaagatg acgatgacaa ggattataaa gatgacgatg
     4021 acaagggagg tggtggtggt ggaggcggtc cgacagagaa tcatcgatgg gtacccagct
     4081 tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
     4141 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4201 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4261 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4321 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4381 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4441 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4501 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4561 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4621 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4681 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4741 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4801 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4861 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4921 gtgctacaga gttcttgaag tggtggccta ac
//
