LOCUS       sequence-342369-        4290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The pE_Cre_lambdaCos plasmid is used to remove the antibiotic 
            resistance cassette from pIT5 integrants..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4290)
  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 4290)
  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: pE_Cre_lambdaCos
FEATURES             Location/Qualifiers
     source          1..4290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(121..225)
                     /gene="bla"
                     /label=AmpR promoter
     RBS             327..335
                     /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             338..1369
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRYGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDYGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             complement(1708..2658)
                     /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      2706..2928
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(3550..4290,1..51,52..120))
                     /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     4173..4192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
       61 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
      181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      241 agtgccacct gcttatcgtg tttgacatgg tgtttagatc tcaatagtat ttagtttaga
      301 tgtagattgt ttagtgcttg atgtactaag gaggtttatg tccaatttac tgaccgtaca
      361 ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca agaacctgat
      421 ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt
      481 ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc
      541 tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag taaaaactat
      601 ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tacgggctgc cacgaccaag
      661 tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg ttgatgccgg
      721 tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg ttcgttcact
      781 catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg ggattgctta
      841 taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata tctcacgtac
      901 tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta gcaccgcagg
      961 tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga tttccgtctc
     1021 tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa atggtgttgc
     1081 agcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt ttgaagcaac
     1141 tcatcgattg atttacggcg ctaaggatga ctatggtcag agatacctgg cctggtctgg
     1201 acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt caataccgga
     1261 gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata tccgtaacct
     1321 ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagt aaccatgggt
     1381 cctccacgca cgttgtgata tgtagatgat aatcattatc actttacggg tcctttccgg
     1441 tgatccgaca ggttacgggg cggcgacctc gcgggttttc gctatttatg aaaattttcc
     1501 ggtttaaggc gtttccgttc ttcttcgtca taacttaatg tttttattta aaataccctc
     1561 tgaaaagaaa ggaaacgaca ggtgctgaaa gcgaggcttt ttgggggtat ggacagtttt
     1621 ccctttgata tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca
     1681 ctgatagata caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc
     1741 tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact
     1801 ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc
     1861 atcgtgtagt gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg
     1921 tattttgtca ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg
     1981 tctatctagt tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac
     2041 caatttcata ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt
     2101 aagcctttta aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag
     2161 gctaatctct atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc
     2221 ataaatcctc atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat
     2281 gaattttttt aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt
     2341 ttcgcttgag aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg
     2401 attcctgatt tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata
     2461 agcattttcc ctactgatgt tcatcatctg aacgtattgg ttataagtga acgataccgt
     2521 ccgttctttc cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat
     2581 tagcttggtt tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa
     2641 aacaactaat tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt
     2701 gagatgggct agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa
     2761 attctgctag acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc
     2821 tagacctttg tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa
     2881 agaataaaaa aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc
     2941 agttccgcag tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc
     3001 ttaaaaccct aaaggcttaa gtagcaccct cgcaagctcg gttgcggccg caatcgggca
     3061 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt
     3121 gacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg
     3181 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc
     3241 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag
     3301 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt
     3361 cagactggct aatgcaccca gtaaggcagc ggtatcatca acggggtctg acgctcagtg
     3421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4261 gttgagatcc agttcgatgt aacccactcg
//
