LOCUS       sequence-342364-        4951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The pAH69 plasmid [Haldimann and Wanner 2001] is the HK022 helper 
            plasmid necessary for genomic integration of the pIT5_KH plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4951)
  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 4951)
  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: pAH69
FEATURES             Location/Qualifiers
     source          1..4951
                     /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)"
     CDS             908..1981
                     /codon_start=1
                     /gene="int"
                     /product="integrase from phage HK022"
                     /label=HK022 integrase
                     /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI
                     TEAIQANIELLSGNRRESLIDRIKGADAITLHAWLDRYETILSERGIRPKTLLDYASKI
                     RAIRRKLPDKPLADISTKEVAAMLNTYVAEGKSASAKLIRSTLVDVFREAIAEGHVATN
                     PVTATRTAKSEVRRSRLTANEYVAIYHAAEPLPIWLRLAMDLAVVTGQRVGDLCRMKWS
                     DINDNHLHIEQSKTGAKLAIPLTLTIDALNISLADTLQQCREASSSETIIASKHHDPLS
                     PKTVSKYFTKARNASGLSFDGNPPTFHELRSLSARLYRNQIGDKFAQRLLGHKSDSMAA
                     RYRDSRGREWDKIEIDK"
     CDS             complement(2118..3068)
                     /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      3116..3338
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(3960..4751,4752..4820))
                     /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     4583..4602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4821..4925)
                     /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 ttcgtagcct tttgaagagg
      901 atcagaaatg ggaagaaggc gaagtcatga gcgccgggat ttacccccta acctttatat
      961 aagaaacaat ggatattact gctacaggga cccaaggacg ggtaaagagt ttggattagg
     1021 ccgagacagg cgaatcgcaa tcactgaagc tatacaggcc aacattgagt tgctatccgg
     1081 gaacaggcgt gagtcactga tagacagaat taaaggcgct gacgcaatca ctcttcatgc
     1141 gtggcttgac cgatatgaaa caatcctcag cgagaggggt atcaggccga aaactctact
     1201 cgactacgcc agcaaaatca gagcaatccg aagaaaattg ccggacaaac cgctcgctga
     1261 catatcaacg aaagaggtgg cagcaatgct aaacacctac gtagcagaag gcaaatcggc
     1321 ttccgcaaaa ttgatcaggt caacccttgt tgacgttttt cgtgaggcaa tagccgaggg
     1381 gcatgtggct acgaatccgg taacagcaac ccgcacagca aaatcagaag taaggcgttc
     1441 aagactgaca gctaatgaat atgtcgcgat ataccatgca gccgaaccgc tcccaatctg
     1501 gctgagactg gcaatggatt tggctgtcgt tacagggcag agagtaggcg atttgtgcag
     1561 aatgaaatgg tcagacataa acgacaacca tcttcacatt gaacagagta aaacaggggc
     1621 taaactcgcc attccactaa cgctaacgat tgacgcgctc aatatctcat tggctgatac
     1681 actacagcaa tgcagggagg ccagcagcag tgaaactata atcgcatcaa agcatcacga
     1741 tccgctttcc ccgaaaacag tatcaaagta ttttacaaag gcgagaaatg catctggact
     1801 ctcatttgat ggaaacccgc caacattcca tgaactgcgt agcctgtcag cgaggctata
     1861 ccggaaccag attggcgata agtttgctca acgtcttctc gggcataaat cagattcaat
     1921 ggcggcgcgg tatagggaca gccgtggacg ggaatgggac aaaattgaaa tcgacaaatg
     1981 attttatttt gactaataat gacctactta ccatgggtat ggacagtttt ccctttgata
     2041 tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca ctgatagata
     2101 caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc tagtgtggtt
     2161 cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact ttgcatgtca
     2221 ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc atcgtgtagt
     2281 gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg tattttgtca
     2341 ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg tctatctagt
     2401 tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac caatttcata
     2461 ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt aagcctttta
     2521 aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag gctaatctct
     2581 atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc ataaatcctc
     2641 atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat gaattttttt
     2701 aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt ttcgcttgag
     2761 aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg attcctgatt
     2821 tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata agcattttcc
     2881 ctactgatgt tcatcatctg aacgtattgg ttataagtga acgataccgt ccgttctttc
     2941 cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat tagcttggtt
     3001 tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa aacaactaat
     3061 tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt gagatgggct
     3121 agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa attctgctag
     3181 acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc tagacctttg
     3241 tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa agaataaaaa
     3301 aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc agttccgcag
     3361 tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc ttaaaaccct
     3421 aaaggcttaa gtagcaccct cgcaagctcg gttgcggccg caatcgggca aatcgctgaa
     3481 tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt gacattcagt
     3541 tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg cgccttttat
     3601 ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc ttctcagggc
     3661 gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag cagttcctgc
     3721 cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt cagactggct
     3781 aatgcaccca gtaaggcagc ggtatcatca acggggtctg acgctcagtg gaacgaaaac
     3841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4561 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4921 ccgcgcacat ttccccgaaa agtgccacct g
//
