LOCUS       sequence-211070-        5623 bp    DNA     circular SYN 15-APR-2025
DEFINITION  integration of loxP site directly upstream of gene start site - 
            contains S. pombe nmt81 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5623)
  AUTHORS   Watson AT1, Garcia V, Bone N, Carr AM, Armstrong J.
  TITLE     Gene tagging and gene replacement using recombinase-mediated 
            cassette exchange in Schizosaccharomyces pombe.
  JOURNAL   Gene. 2008 Jan 15;407(1-2):63-74
  PUBMED    18054176
REFERENCE   2  (bases 1 to 5623)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene."; 
            date: "2008-01-15"; volume: "407(1-2)"; pages: "63-7"
COMMENT     Sequence Label: pAW34
FEATURES             Location/Qualifiers
     source          1..5623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..24)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6..24)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(31..48)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(31..47)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(40..62)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      188..643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(275..294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     485..506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(710..729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     829..851
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(889..907)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        975..1079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1080..1148,1149..1940)
                     /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     complement(1298..1317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2111..2699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2600..2619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2853..2870
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2987..3008
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3023..3028,3029..3046,3047..3053)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3061..3077
                     /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)."
     primer_bind     3066..3088
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3085..3101
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3085..3101
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3119..3137
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3119..3136
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    complement(3154..3187)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3721..4515
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVSGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     protein_bind    complement(5570..5603)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
       61 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      121 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      181 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      241 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      301 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      361 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      421 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      481 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      541 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      601 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
      661 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
      721 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
      781 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
      841 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
      901 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
      961 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1021 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1081 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1141 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1201 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1261 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1321 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1381 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1441 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1501 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1561 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1621 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1681 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1741 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1801 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1861 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1921 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1981 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2221 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2881 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2941 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     3001 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     3061 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
     3121 ttaggtgaca ctatagaata ctcaagcttg gacataactt cgtatagcat acattatacg
     3181 aagttatctg cagagctaca aatcccactg gctatatgta tgcatttgtg ttaaaaaagt
     3241 ttgtatagat tatttaatct actcagcatt ctttctctaa ataggaattt gttacttaat
     3301 ggagaaaaaa atgtttcgat ttacctagtg tatttgtttg tatactcacg tttaatttca
     3361 aacatccatt ctatcttgtg taatttttgg catggtgaaa aagataatca gccttataat
     3421 ctttacaaaa gtaagaaatt ctgtaaataa gccttaatgc ccttgcttta aattaaaatg
     3481 gttctttttc atgataatgt ttgcactttg tgaatatatt ttagatagtt ctgtgaggta
     3541 taattaagat gttttagaga cttatacaat tttgtcttta taaattctta attgatttta
     3601 ccatcccagt ttaactatgc ttcgtcggca tctctgcaca tgtcgtgttt tcttaccgta
     3661 ttgtcctacc aagaacctct tttttgcttg gatcgaaatt aaaggtttaa aagcaaagtt
     3721 atggatgcta gagtatttca aagctattca gctagagctg aggggatgaa aaatcccatt
     3781 gccaaggaat tgttggcttt gatggaagaa aagcaaagca acttgtcagt cgcggtcgat
     3841 ttgacgaaga aatccgaaat cttagaattg gtagataaaa ttggacccta tgtctgtgtt
     3901 atcaagacac atattgacgt tgtcgaggat ttcgaccagg atatggtaga aaaactggtg
     3961 gccttaggta aaaagcatcg ttttcttatc tttgaggatc gcaaattcgc agacattgga
     4021 aataccgtca agctacaata tgcatctggt gtgtacaaaa ttgcttcttg ggctcatatc
     4081 acaaattgcc atacagtgtc aggcgagggt attatacaag gcctcaaaga agttggttta
     4141 cctttgggac gtggtctctt gcttttggct gaaatgtctt ccaaaggctc tttggctact
     4201 ggttcctaca cagagaaaac cttagaatgg tttgagaagc ataccgattt ttgctttggc
     4261 tttatagctg gtcgtcgatt tcctaacctt caaagcgact acataactat gtcccctggt
     4321 atcggcttgg atgttaaagg agacgggctg ggacagcaat atcgtactcc tgaagaagtg
     4381 attgtaaact gcggtagcga tatcatcatt gttggtcgtg gagtctatgg agctggtcgt
     4441 aatcctgttg tcgaagccaa gagatataga gaagctggtt ggaaggcata tcagcaaaga
     4501 ctttctcagc attaaaaaaa gactaatgta aaattttttt ggttggttat tgaaaaagtc
     4561 gatgccttgt ttgcgtttgt tttcctaggc gttttatgtc agaaggcatt tagaattagt
     4621 atacaagtac tctttggtaa aattttatgt agcgactaaa atattaacta ttatagataa
     4681 acaccttggg aataaaaagt aatttgctat agtaatttat taaacatgct cctacaacat
     4741 taccacaatc ttttctcttg gattgacatt gaataagaaa agagtgaatt tttttagact
     4801 tgtaatgata actatgtaca aagccaatga aagatgtatg tagatgaatg taaaatacca
     4861 tgtagacaaa caagataaaa cttggttata aacattggtg ttggaacaga ataaattaga
     4921 tgtcaaaaag tttcgtcaat atcacaagcc tgcagagatc tgttgaaccg aaattgcaga
     4981 tactgatata tgaaaataat acccacaatt ttgggaatag cgcaagcctc aatttaaaca
     5041 ataggtgagg acacatgata atgacctcaa tgattgttag aagaaaagag cctcattaca
     5101 aaatcgaaaa atgaatggtt gggtacaagt ttccaaaaca tggtaaagtg gactttgcgt
     5161 atgagacgta aatagaaaaa aacacttgtt atatgttttc tagaattatt gttgtctctt
     5221 tatggttgga tgatgcaaaa tagtaatttc ggttagttgc tgtaaaacac cacgagacaa
     5281 atagatatgg atatttatta aatcaggaaa aacgtaactc tcggctactg gatggttcag
     5341 tcacccaacg attactgggg agagaaaaca gggcaaaagc aaagcttaaa ggaatccgat
     5401 tgtcattcgg caatgtgcag cgaaactaaa aaccggataa tggacctgtt aatcgaaaca
     5461 ttgaagatgg aagaggaatc ctggcatatc atcaattgaa taagttgaat taattatttc
     5521 aatctcattc tcactttctg acttatagtc gctttgttaa atcagatcta taacttcgta
     5581 tagcatacat tatacgaagt tatcgggcgg cgggggtggt gaa
//
