LOCUS       sequence-342319-        4861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The D145 helper plasmid necessary for genomic integration of the 
            pIT5_KD plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4861)
  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 4861)
  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_D
FEATURES             Location/Qualifiers
     source          1..4861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(71..784)
                     /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             873..881
                     /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             893..915
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(2037..2987)
                     /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      3035..3257
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(3879..4670,4671..4739))
                     /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     4502..4521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4740..4844)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctgc atcgatgaag attcttgctc aattgttatc agctatgcgc cgaccagaac
       61 accttgccga tcagccaaac gtctcttcag gccactgact agcgataact ttccccacaa
      121 cggaacaact ctcattgcat gggatcattg ggtactgtgg gtttagtggt tgtaaaaaca
      181 cctgaccgct atccctgatc agtttcttga aggtaaactc atcaccccca agtctggcta
      241 tgcagaaatc acctggctca acagcctgct cagggtcaac gagaattaac attccgtcag
      301 gaaagcttgg cttggagcct gttggtgcgg tcatggaatt accttcaacc tcaagccaga
      361 atgcagaatc actggctttt ttggttgtgc ttacccatct ctccgcatca cctttggtaa
      421 aggttctaag cttaggtgag aacatccctg cctgaacatg agaaaaaaca gggtactcat
      481 actcacttct aagtgacggc tgcatactaa ccgcttcata catctcgtag atttctctgg
      541 cgattgaagg gctaaattct tcaacgctaa ctttgagaat ttttgtaagc aatgcggcgt
      601 tataagcatt taatgcattg atgccattaa ataaagcacc aacgcctgac tgccccatcc
      661 ccatcttgtc tgcgacagat tcctgggata agccaagttc atttttcttt ttttcataaa
      721 ttgctttaag gcgacgtgcg tcctcaagct gctcttgtgt taatggtttc ttttttgtgc
      781 tcatacgtta aatctatcac cgcaagggat aaatatctaa caccgtgcgt gttgactatt
      841 ttacctctgg cggtgataat ggttgcatgt actaaggagg ttgtatgaat tctttgttta
      901 actttaagaa ggagatatac atatgtctat taaaaagctc gatgatggtc gttatgaagt
      961 ggatgtcaga ccgcagggtg cagatggaaa acgtatcagg cggaaattta aaactaaagg
     1021 tgaagctcaa gcattcgaac gtcatgtact ggttaactac cacaacaaag agtggctgga
     1081 gaagccagcc gaccgccgaa ctcttacaga gttgttaggc agatggtgga tatatcacgg
     1141 aaaatcacat gagcgtggag atattgaacg ggggcgtttg acgacaataa tcgccaaatt
     1201 tgcagaaatg ggagtgtcca gagctgacca gctaacaaag aaaacgataa ctgattatcg
     1261 cgttgtaatg atgaacgatg gtctaaaacc agccagcgta aatcggcatc tggcaataat
     1321 gagcgggatg ttcaccaagt taattgatgc cggtgaatac cattctcaca acccgttccg
     1381 tgaggttaag cgattacgtg aagctgttac agaaatggct tttttgtcca gtgaagagat
     1441 tacacggctg ttatccatgc tcgatggtga tgaattaaat gcgactctgg tctgcctttc
     1501 tactggtgga cgctggagtg aagtgtctaa tttaaaagct gaacacatca ttaaccagat
     1561 ggttacgttt atgaaaacta aaaacggaaa gcgcaggaca attcccgttt cgcaggacct
     1621 gattaaacgg atcaagacca aaaattcagg caggcttttt aatgccagtt actacaaagt
     1681 gcgcaacgct ctcagggaag taaaacccga tttacttgac ggacaggcag tgcatgtttt
     1741 gaggcataca tttgccacac attttataat gaatggaggt aacataatca cattgcagcg
     1801 catcctgggt cattctcaca ttcagcaaac tatgacctac gcacactttg caccggattt
     1861 cttacaagat gcagtgactc ttaacccggt gtcaggaatg tccataatgc gtccataatt
     1921 ttattttgac catgggtatg gacagttttc cctttgatat gtaacggtga acagttgttc
     1981 tacttttgtt tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag
     2041 atccttccgt atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat
     2101 gagaacgaac cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa
     2161 ctggtgagct gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttatgt
     2221 aggtaggaat ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg
     2281 ttctcaagtt cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta
     2341 tcagtcgggc ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct
     2401 ttacttattg gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca
     2461 agcattaaca tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc
     2521 ttttgtgtta gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa
     2581 gacttaacat gttccagatt atattttatg aattttttta actggaaaag ataaggcaat
     2641 atctcttcac taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac
     2701 tggaaaatct caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc
     2761 tctctggttg ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga
     2821 acgtattggt tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg
     2881 gggttgagta gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag
     2941 cgactaatcg ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg
     3001 tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc
     3061 cttttccttt gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta
     3121 aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt
     3181 caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc
     3241 agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa
     3301 acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc
     3361 gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat attccttttg tctccgacca
     3421 tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg
     3481 cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac
     3541 ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat
     3601 gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac
     3661 cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg
     3721 gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3781 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3841 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3901 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3961 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4021 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4081 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4141 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4201 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4261 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4321 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4381 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4441 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4501 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4561 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4621 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4681 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4741 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4801 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4861 g
//
