LOCUS       sequence-497166-        5408 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  P4Lacn.2 tagging casette for WTC848 with Hygromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5408)
  TITLE     Stelling Lab Plasmids 2025
REFERENCE   2  (bases 1 to 5408)
  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: P2879
FEATURES             Location/Qualifiers
     source          1..5408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     167..186
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     400..422
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     414..431
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     415..431
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     438..457
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        438..456
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            516..2091
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        516..859
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             860..1888
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      1894..2091
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    2722..2738
                     /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    2751..2767
                     /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    2843..2859
                     /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    2864..2880
                     /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             2901..2972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     promoter        complement(3035..3053)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3074..3090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3074..3090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3087..3109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3098..3114
                     /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(3122..3128,3129..3146,3147..3152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3167..3188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3305..3322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3476..4064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3556..3575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4235..5026,5027..5095))
                     /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     4858..4877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5096..5200)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5226..5408,1..273))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5363..5384)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
       61 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
      121 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
      181 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
      241 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
      301 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      361 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      421 cgacggccag tgaattgtaa tacgactcac tatagggcga attggagctc gatcctcgag
      481 agcttgcctt gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
      541 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
      601 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
      661 gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg aaacgctccc ctcacagacg
      721 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
      781 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
      841 catccgaaca taaacaacca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
      901 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
      961 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     1021 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     1081 gattccggaa gtgcttgaca ttggggagtt cagcgagagc ctgacctatt gcatctcccg
     1141 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     1201 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     1261 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     1321 gattgctgat ccccacgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     1381 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     1441 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     1501 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     1561 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     1621 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     1681 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     1741 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     1801 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     1861 cagcactcgt ccgagggcaa aggaataatc agtactgaca ataaaaagat tcttgttttc
     1921 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     1981 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     2041 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     2101 cgatactaac gccgccatcc agtgtttaaa cgaactagtg cggccgccag ttcgagttta
     2161 tcattatcaa tactgccatt tcaaagaata cgtaaataat taatagtagt gattttccta
     2221 actttattta gtcaaaaaat tagcctatat aacatcgtag gtgtctgggt gaacagttta
     2281 ttcctggcat ccactaattt taattctgct gtaacccgta catgcccaaa atagggggcg
     2341 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca
     2401 ctaaatataa tggagcccgc tttttaagct ggcatccact aaaaaaaaga atcccagcac
     2461 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac
     2521 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct
     2581 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt
     2641 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa
     2701 ccagttccct gaaattatta attgtgagcg gataacaaag tataaataga ttgtgagcgg
     2761 ataacaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt
     2821 ttttttagtt ttaaaacacc aattgtgagc ggataacaat taattgtgag cggataacaa
     2881 ttacacacat aaacaaaatg gattataaag atgacgatga caaggattat aaagatgacg
     2941 atgacaagga ttataaagat gacgatgaca agggaggtgg tggtggtgga ggcggtccga
     3001 cagagaatca tcgatgggta cccagctttt gttcccttta gtgagggtta atttcgagct
     3061 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3121 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3181 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3241 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3301 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3361 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3661 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3721 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3781 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3841 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3901 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3961 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4021 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4081 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4141 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4201 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4321 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4381 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4801 cattctgaga atagtgtatg cggcgaccga gttgctcttg tccggcgtca atacgggata
     4861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5161 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5221 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5281 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5341 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5401 ctccaacg
//
