LOCUS       sequence-357432-        4527 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial genomic integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4527)
  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 4527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_KW
FEATURES             Location/Qualifiers
     source          1..4527
                     /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)."
     terminator      complement(3233..3479)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(3570..3592)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3743..3761
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      3802..3888
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3985..4071
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4168..4254
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4351..4437
                     /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 ccatggtggt cgatgtggac gctatgtgga
     3001 cagtggttga tataaatcca tttatatcat caggttaggt gcttttttgt gacaccatcc
     3061 ctgtcttccc ccacatgatg tgggggtttt ttttatcaat cacttacatc ggtttccgta
     3121 agaaaacagt aacttaagaa aacctataag ggactggaag ggctacaaag ttctgagagt
     3181 gtggacacta tgtggacact cagaagggaa atgctagctt cttcgtctgt ttctactggt
     3241 attggcacaa acctgattcc aatttgagca aggctatgtg ccatctcgat actcgttctt
     3301 aactcaacag aagatgcttt gtgcatacag cccctcgttt attatttatc tcctcagcca
     3361 gccgctgtgc tttcagtgga tttcggataa cagaaaggcc gggaaatacc cagcctcgct
     3421 ttgtaacgga gtagagacga aagtgattgc gcctacccgg atattatcgt gaggatgcgt
     3481 catcgccatt aattcactga tcagtgataa gctgtcaaac atgagaattg atccggctgc
     3541 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3601 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3661 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatgct
     3721 gcacatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
     3781 aattcccaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     3841 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     3901 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     3961 gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4021 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     4081 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     4141 actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     4201 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     4261 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     4321 taaactgcca ggaattaatt ccccaggcat caaataaaac gaaaggctca gtcgaaagac
     4381 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     4441 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     4501 ccataaactg ccaggaattg gggatcg
//
