LOCUS       sequence-100282-        4361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Phagemid expressing streptomycin/spectinomycin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4361)
  AUTHORS   Firnberg E, Ostermeier M
  TITLE     PFunkel: efficient, expansive, user-defined mutagenesis.
  JOURNAL   PLoS One. 2012;7(12):e52031. doi: 10.1371/journal.pone.0052031. Epub
            2012 Dec 17.
  PUBMED    23284860
REFERENCE   2  (bases 1 to 4361)
  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: "PLoS One.";
            date: "2012"; volume: "7(12):e52031. doi"; pages: " 
            10.1371/journal.pone.0052031. Epub 2012 Dec 17"
COMMENT     Sequence Label: pSkunk3-BLA
FEATURES             Location/Qualifiers
     source          1..4361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..79
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     71..88
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     72..88
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     95..114
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        95..113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(433..438,439..454,455..461)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    469..485
                     /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     474..496
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             join(508..576,577..1368)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(726..745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(1535..2323)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2324..2426)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2818..2835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2952..3496)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3032..3051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3572..3590
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3628..3650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3750..3769
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3836..4291)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3973..3994)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4185..4204
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
       61 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt
      121 ctggcgaatc ctctgaccag ccagaaaacg acctttctgt ggtgaaaccg gatgctgcaa
      181 ttcagagcgc cagcaagtgg gggacagcag aagacctgac cgccgcagag tggatgtttg
      241 acatggtgaa gactatcgca ccatcagcca gaaaaccgaa ttttgctggg tgggctaacg
      301 atatccgcct gatgcgtgaa cgtgacggac gtaaccaccg cgacatgtgt gtgctgttcc
      361 gctgggcatg ccaggacaac ttctggtccg gtaacgtgct gagcccggcc aagcttactc
      421 cccatccccc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat
      481 aacaatttca cacaggagga aggatccatg agtattcaac atttccgtgt cgcccttatt
      541 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      601 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      661 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
      721 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
      781 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      841 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
      901 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      961 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1021 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     1081 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1141 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1201 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     1261 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1321 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaaac tagttagtaa
     1381 gcttatgcat tcgaccggcc gacgcgctgg gctacgtctt gctggcgttc ggggtcgaat
     1441 ttgctttcga atttctgcca ttcatccgct tattatcact tattcaggcg tagcaaccag
     1501 gcgtttaagg gcaccaataa ctgccttaaa aaaatttgcc gactaccttg gtgatctcgc
     1561 ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt
     1621 cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc
     1681 gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt
     1741 accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg
     1801 agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat
     1861 caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca
     1921 gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat
     1981 tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt
     2041 cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag
     2101 ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg
     2161 tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc
     2221 cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct
     2281 ctgatagttg agtcgatact tcggcgatca ccgcttccct cattttagct tccttagctc
     2341 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     2401 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccagggct
     2461 tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct tccgtcacag
     2521 gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttactgatt tagtgtatga
     2581 tggtgttttt gaggtgctcc agtggcttct gtttctatca gctgtccctc ctgttcagct
     2641 actgacgggg tggtgcgtaa cggcaaaagc accgccggac atcagcgcta gcggagtgta
     2701 tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag
     2761 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
     2821 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
     2881 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
     2941 gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca
     3001 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc cctggcggct
     3061 ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc
     3121 cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg
     3181 gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt
     3241 cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga
     3301 tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt
     3361 tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac
     3421 cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag
     3481 accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct agagctaaga
     3541 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     3601 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3661 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3721 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3781 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     3841 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3901 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3961 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4021 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4081 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4141 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4201 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4261 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     4321 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc a
//
