LOCUS       sequence-363986-        4602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial genomic integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4602)
  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 4602)
  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: pIT5_KO
FEATURES             Location/Qualifiers
     source          1..4602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             142..447
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     326..345
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     rep_origin      477..1065
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     966..985
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1125..1145
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1200..1227
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1276..1307)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     protein_bind    1383..1416
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1580..1599)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1591..2406
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1664..1683)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2528..2916
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    2931..2964
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2977..3191
                     /gene="attP"
                     /label=phage 186 attP
                     /bound_moiety="phage 186 integrase"
                     /note="attachment site of phage 186"
     terminator      complement(3308..3554)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(3645..3667)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3818..3836
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      3877..3963
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4060..4146
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4243..4329
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4426..4512
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gaattcgagc tcggtacccg gggatcctct agagggctta ctaaaagcca gataacagta
       61 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
      121 tatgtcaaaa agaggtatgc tatgcagttt aaggtttaca cctataaaag agagagccgt
      181 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
      241 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
      301 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
      361 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
      421 attaacctga tgttctgggg aatataataa cccgtagaaa agatcaaaag atcttcttga
      481 gatccttttt ttctgcgcgt aatctgctac ttgcaaacaa aaaaaccacc gctaccagcg
      541 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggctttagc
      601 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
      661 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      721 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      781 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      841 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      901 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      961 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1021 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1081 gcctttttac ggttcctggc cttttgctgg ccttttgctc acattactag tagcggccgc
     1141 tgcaggcatg cctcgagatg catggcgcct aacctaaact gacaggcatc aaattaagca
     1201 gaaggccatc ctgacggatg gcctttttgc gtttcgaaca attgaaaaaa cctcgcgcct
     1261 tacctgttga gtaatagtca aaagcctccg gtcggaggct tttgactttc tgcttactga
     1321 atttcggtgg tgccgttaat taaccggtgg gccctcatga taataatggt ttcttagacg
     1381 tctaccgttc gtataatgta tgctatacga agttatccta ggcgacaatg taactactag
     1441 agctgatcct tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtgtctcaa
     1501 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     1561 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     1621 tcccgtccgc gcttaaactc caacatggac gctgatttat atgggtataa atgggctcgc
     1681 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     1741 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     1801 cgtctcaact ggctgacgga gtttatgcct ctcccgacca tcaagcattt tatccgtact
     1861 cctgatgatg cgtggttact caccaccgcg attcctggga aaacagcctt ccaggtatta
     1921 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggccgtgtt cctgcgccgg
     1981 ttacattcga ttcctgtttg taattgtcct tttaacagcg atcgtgtatt tcgtcttgct
     2041 caggcgcaat cacgcatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     2101 aatggctggc ctgttgaaca agtctggaaa gaaatgcaca agctcttgcc attctcaccg
     2161 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     2221 ttaataggtt gtattgatgt tggacgggtc ggaatcgcag accgttacca ggaccttgcc
     2281 attctttgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     2341 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     2401 ttctaataat actagcagaa atcatcctta gcgaaagcta aggatttttt ttatctgatt
     2461 accgcctttg agtgagcgtc gacctagtgc ggccgcaaga tccggccacg atgcgtccgg
     2521 cgtagaggat ctgaagatca gcagttcaac ctgttgatag tacgtactaa gctctcatgt
     2581 ttcacgtact aagctctcat gtttaacgta ctaagctctc atgtttaacg aactaaaccc
     2641 tcatggctaa cgtactaagc tctcatggct aacgtactaa gctctcatgt ttcacgtact
     2701 aagctctcat gtttgaacaa taaaattaat ataaatcagc aacttaaata gcctctaagg
     2761 ttttaagttt tataagaaaa aaaagaatat ataaggcttt taaagctttt aaggtttaac
     2821 ggttgtggac aacaagccag ggatgtaacg cactgagaag cccttagagc ctctcaaagc
     2881 aattttgagt gacacaggaa cacttaacgg ctgacatggg aattaggaag ataacttcgt
     2941 ataatgtatg ctatacgaac ggtacatatg ccatgggcga tggttctgag taacagataa
     3001 tagaatggcg ataaagtggc ggtagaaatg gcgaataata ggtaattatt ggcaaacaat
     3061 ggcaatctat gtcaatgata aataacgcat actattgatt ttcggttgtt cttgtaggaa
     3121 ctcataatcg cttggtcgct ggttcaagtc cagcaggggc caccaaattt aagtagtaaa
     3181 tacatacagt taggccactc tctcgagtgg cctttttgtt gcctcgtttt cgagtggcga
     3241 tagaatggcg gtggwttttt taccgccact ttcctgagga gtgcatagct agcttcttcg
     3301 tctgtttcta ctggtattgg cacaaacctg attccaattt gagcaaggct atgtgccatc
     3361 tcgatactcg ttcttaactc aacagaagat gctttgtgca tacagcccct cgtttattat
     3421 ttatctcctc agccagccgc tgtgctttca gtggatttcg gataacagaa aggccgggaa
     3481 atacccagcc tcgctttgta acggagtaga gacgaaagtg attgcgccta cccggatatt
     3541 atcgtgagga tgcgtcatcg ccattaattc actgatcagt gataagctgt caaacatgag
     3601 aattgatccg gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3661 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     3721 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     3781 agcggagtgt atgctgcaca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     3841 tcgtcttcaa gaattaattc ccaattcccc aggcatcaaa taaaacgaaa ggctcagtcg
     3901 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3961 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     4021 cgcccgccat aaactgccag gaattaattc cccaggcatc aaataaaacg aaaggctcag
     4081 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     4141 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     4201 ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa acgaaaggct
     4261 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     4321 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     4381 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
     4441 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4501 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     4561 cgggcaggac gcccgccata aactgccagg aattggggat cg
//
