LOCUS       sequence-342368-        4841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The Wphi helper plasmid necessary for genomic integration of the 
            pIT5_KW plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4841)
  AUTHORS   Hao N, Chen Q, Dodd IB, Shearwin KE
  TITLE     The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome 
            Integration in E. coli.
  JOURNAL   ACS Synth Biol. 2021 Jun 30. doi: 10.1021/acssynbio.1c00215.
  PUBMED    34190535
REFERENCE   2  (bases 1 to 4841)
  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: "ACS Synth 
            Biol. 2021 Jun 30. doi: 10.1021/acssynbio.1c00215."
COMMENT     Sequence Label: pHelper_W
FEATURES             Location/Qualifiers
     source          1..4841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(62..775)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             864..872
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             884..906
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(2008..2958)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3006..3228
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(3850..4641,4642..4710))
                     /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     4473..4492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4711..4815)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgatgaa gattcttgct caattgttat cagctatgcg ccgaccagaa caccttgccg
       61 atcagccaaa cgtctcttca ggccactgac tagcgataac tttccccaca acggaacaac
      121 tctcattgca tgggatcatt gggtactgtg ggtttagtgg ttgtaaaaac acctgaccgc
      181 tatccctgat cagtttcttg aaggtaaact catcaccccc aagtctggct atgcagaaat
      241 cacctggctc aacagcctgc tcagggtcaa cgagaattaa cattccgtca ggaaagcttg
      301 gcttggagcc tgttggtgcg gtcatggaat taccttcaac ctcaagccag aatgcagaat
      361 cactggcttt tttggttgtg cttacccatc tctccgcatc acctttggta aaggttctaa
      421 gcttaggtga gaacatccct gcctgaacat gagaaaaaac agggtactca tactcacttc
      481 taagtgacgg ctgcatacta accgcttcat acatctcgta gatttctctg gcgattgaag
      541 ggctaaattc ttcaacgcta actttgagaa tttttgtaag caatgcggcg ttataagcat
      601 ttaatgcatt gatgccatta aataaagcac caacgcctga ctgccccatc cccatcttgt
      661 ctgcgacaga ttcctgggat aagccaagtt catttttctt tttttcataa attgctttaa
      721 ggcgacgtgc gtcctcaagc tgctcttgtg ttaatggttt cttttttgtg ctcatacgtt
      781 aaatctatca ccgcaaggga taaatatcta acaccgtgcg tgttgactat tttacctctg
      841 gcggtgataa tggttgcatg tactaaggag gttgtatgaa ttctttgttt aactttaaga
      901 aggagatata catatgtcaa ttaagaagct cgatgatgga cgctatgaag tggacattag
      961 acctcgcggt cgcgacggaa aacgcatccg caggaaattt gaaagaaaag ctgaggctgt
     1021 agcatttgag cgatacacaa tcgcctacgc cagccagaaa gaatgggcag gtcagcgagc
     1081 agatcgccga actttgagtg agttgctgga catctggtgg aaatatcacg ggcaaaacca
     1141 cgagcatgga acaaaagagt ttaatcatct gctcaaaacc atcagcggca taggtgatat
     1201 accagtgagc cggatgagca aaagagcttt gatggattat cgttccatgc gactacgtga
     1261 tggtatcagt gccgcaacga taaaccgtga catgtaccga ttatccggca tgttcacaaa
     1321 attaattcaa ttggatgaat tttccgggca acacccaatt cacggactgc cgccactggc
     1381 ggaggccaac cctgaaatga cgttcctgga aaaagcagaa atcgaaaaac tgttaaatgt
     1441 tttggctggt gatgacttac ttgtcgcact tttatgtctg agcactggag gaagatggac
     1501 ggaagttgcc acgctaaaac cagcacagat tacaaattgc agggttacct tcctgaaaac
     1561 caaaaacggt aaaaagcgaa ccgtgccgat ttctgaggaa ctggagaaaa aagttaaaga
     1621 ggaggccagc gctaaattat tcaaagttga ttatgagaag ttttgcggga ttttacgcag
     1681 agtgaagcca gatataccac ccaatcaggc aacccacatc ctgcggcata cattcgcaag
     1741 ccatttcatg atgaatgggg gcaatataat cgcactgcaa cagattctgg gacatgcgag
     1801 cattcagcag acgatggcct atgcgcacct tgcgcctgac tacctgcaaa atgccgtcgc
     1861 gctgaatcct ctaaaaggcg gagtgacgtt ataagctagc ccatgggtat ggacagtttt
     1921 ccctttgata tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca
     1981 ctgatagata caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc
     2041 tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact
     2101 ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc
     2161 atcgtgtagt gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg
     2221 tattttgtca ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg
     2281 tctatctagt tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac
     2341 caatttcata ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt
     2401 aagcctttta aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag
     2461 gctaatctct atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc
     2521 ataaatcctc atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat
     2581 gaattttttt aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt
     2641 ttcgcttgag aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg
     2701 attcctgatt tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata
     2761 agcattttcc ctactgatgt tcatcatctg aacgtattgg ttataagtga acgataccgt
     2821 ccgttctttc cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat
     2881 tagcttggtt tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa
     2941 aacaactaat tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt
     3001 gagatgggct agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa
     3061 attctgctag acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc
     3121 tagacctttg tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa
     3181 agaataaaaa aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc
     3241 agttccgcag tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc
     3301 ttaaaaccct aaaggcttaa gtagcaccct cgcaagctcg gttgcggccg caatcgggca
     3361 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt
     3421 gacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg
     3481 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc
     3541 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag
     3601 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt
     3661 cagactggct aatgcaccca gtaaggcagc ggtatcatca acggggtctg acgctcagtg
     3721 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3781 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3841 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3901 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3961 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4021 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4081 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4141 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4201 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4261 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4321 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4381 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4441 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4501 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4561 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4621 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4681 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4741 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4801 aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
