LOCUS       sequence-342367-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The 186 helper plasmid necessary for genomic integration of the 
            pIT5_KO plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  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 4887)
  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_O
FEATURES             Location/Qualifiers
     source          1..4887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(284..1075,1076..1144))
                     /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     907..926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1145..1249)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(1337..2050)
                     /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             2139..2147
                     /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)"
     CDS             2180..3190
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage 186"
                     /label=phage 186 integrase
                     /translation="MTVRKNPAGGWICELYPNGAKGKRIRKKFATKGEALAFEQYTVQN
                     PWQEEKEDRRTLKGLVDSWYSAHGITLKDGLKRQLAMHHAFECMGEPLARDFDAQMFSR
                     YREKRLKGEYARSNRVKEVSPRTLNLELAYFRAVFNELNRLGEWKGENPLKNMRPFRTE
                     EMEMTWLTHDQISQLLGECNRHDHPDLETVVRICLATGARWSEAESLRKSQLAKYKITY
                     TNTKGRKNRTVPISKELYESLPDDKKGRLFSDCYGAFRSALERTGIELPAGQLTHVLRH
                     TFASHFMMNGGNILVLQRVLGHTDIKMTMRYAHFAPDHLEDAVKLNPLVHITNSK"
     CDS             complement(3329..4279)
                     /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      4327..4549
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 gctatgtggt gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt
       61 ctgaccactt cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg
      121 cagcggtatc atcaacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      181 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      241 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      301 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
      361 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
      421 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
      481 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
      541 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      601 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      661 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      721 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
      781 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
      841 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
      901 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
      961 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1021 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1081 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1141 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1201 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1261 gaaaagtgcc acctgcatcg atgaagattc ttgctcaatt gttatcagct atgcgccgac
     1321 cagaacacct tgccgatcag ccaaacgtct cttcaggcca ctgactagcg ataactttcc
     1381 ccacaacgga acaactctca ttgcatggga tcattgggta ctgtgggttt agtggttgta
     1441 aaaacacctg accgctatcc ctgatcagtt tcttgaaggt aaactcatca cccccaagtc
     1501 tggctatgca gaaatcacct ggctcaacag cctgctcagg gtcaacgaga attaacattc
     1561 cgtcaggaaa gcttggcttg gagcctgttg gtgcggtcat ggaattacct tcaacctcaa
     1621 gccagaatgc agaatcactg gcttttttgg ttgtgcttac ccatctctcc gcatcacctt
     1681 tggtaaaggt tctaagctta ggtgagaaca tccctgcctg aacatgagaa aaaacagggt
     1741 actcatactc acttctaagt gacggctgca tactaaccgc ttcatacatc tcgtagattt
     1801 ctctggcgat tgaagggcta aattcttcaa cgctaacttt gagaattttt gtaagcaatg
     1861 cggcgttata agcatttaat gcattgatgc cattaaataa agcaccaacg cctgactgcc
     1921 ccatccccat cttgtctgcg acagattcct gggataagcc aagttcattt ttcttttttt
     1981 cataaattgc tttaaggcga cgtgcgtcct caagctgctc ttgtgttaat ggtttctttt
     2041 ttgtgctcat acgttaaatc tatcaccgca agggataaat atctaacacc gtgcgtgttg
     2101 actattttac ctctggcggt gataatggtt gcatgtacta aggaggttgt atgaattcct
     2161 ttaagaagga gatatacata tgaccgtccg taaaaatccg gctggcggtt ggatttgtga
     2221 actctaccca aacggtgcaa aaggcaaacg tatcagaaag aaattcgcta ctaaaggcga
     2281 ggctctggcg tttgagcagt acaccgttca aaacccgtgg caggaagaaa aggaagacag
     2341 gcgcacgtta aaagggctgg ttgattcatg gtatagcgct catggcatta cactgaaaga
     2401 tggtttgaaa cgccagttag ccatgcacca tgcttttgag tgtatgggcg aaccactcgc
     2461 acgcgatttc gatgcgcaga tgttttcccg ctaccgagaa aaacggttaa aaggtgagta
     2521 tgcccgttca aacagagtga aagaggtatc gcctcgcacg cttaatcttg agctggccta
     2581 cttccgggcg gtgttcaatg agctaaaccg cctcggagaa tggaagggtg aaaacccact
     2641 aaaaaatatg cgcccattcc gcacagaaga aatggaaatg acttggctaa ctcacgacca
     2701 aatttcgcaa ctgctcggag agtgtaaccg acatgaccac cctgatttag aaaccgtggt
     2761 aagaatctgt ctcgcaactg gcgcacggtg gtctgaggcc gagagtctga gaaaaagcca
     2821 actcgcgaaa tacaaaatca catacaccaa cacgaaaggt agaaaaaatc gcaccgtccc
     2881 aatcagcaaa gagctctatg agtctctgcc tgatgataaa aagggccggt tgttcagtga
     2941 ttgttatggc gcgttccggt cagctttgga aagaacaggt atcgaactac cggcaggaca
     3001 acttacccac gttttacgtc acaccttcgc tagtcacttt atgatgaatg gcggtaatat
     3061 tttggtgttg cagcgcgtac tcggccatac agatataaaa atgaccatgc gatatgcgca
     3121 ctttgcaccc gaccatttag aggatgcagt taaactaaat cctttagttc acattactaa
     3181 cagcaaataa gttatggagg ttgtatgtca gttcggctag cccatgggta tggacagttt
     3241 tccctttgat atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc
     3301 actgatagat acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct
     3361 ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatacttac
     3421 tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag
     3481 catcgtgtag tgtttttctt agtccgttat gtaggtagga atctgatgta atggttgttg
     3541 gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt
     3601 gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca
     3661 ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt
     3721 taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa
     3781 ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact
     3841 cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta
     3901 tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact aattctaatt
     3961 tttcgcttga gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag
     4021 gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat
     4081 aagcattttc cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg
     4141 tccgttcttt ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa
     4201 ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga
     4261 aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat
     4321 tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta
     4381 aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg
     4441 ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga
     4501 aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt
     4561 cagttccgca gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac
     4621 cttaaaaccc taaaggctta agtagcaccc tcgcaagctc ggttgcggcc gcaatcgggc
     4681 aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg
     4741 tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag
     4801 gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg
     4861 cttctcaggg cgttttatgg cgggtct
//
