LOCUS       sequence-383676-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses I-SceI homing endonuclease from the T7 promoter containing
            an additional methionine-histidine sequence at the amino terminus 
            and a K122I mutation that results in nicking activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  AUTHORS   Niu Y, Tenney K, Li H, Gimble FS
  TITLE     Engineering variants of the I-SceI homing endonuclease with 
            strand-specific and site-specific DNA-nicking activity.
  JOURNAL   J Mol Biol. 2008 Sep 26;382(1):188-202. doi: 
            10.1016/j.jmb.2008.07.010. Epub 2008 Jul 11.
  PUBMED    18644379
REFERENCE   2  (bases 1 to 6371)
  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"; doi: 
            "10.1016/j.jmb.2008.07.010"; journalName: "J Mol Biol"; date: 
            "2008-09-26- 26"; volume: "382"; issue: "1"; pages: "188-202"
COMMENT     Sequence Label: pET15b I-SceI (Ctermhis) K122I
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1980..1999
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2019..2096)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2302..2321
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2405..2424
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2405..2423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2424..2448
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             2463..2485
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3206..3223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3277..3295)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3291..3338
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3513..3518,3519..3535,3536..3541))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3568..3586)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3654..3758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3759..3827,3828..4619)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3977..3996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4790..5378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5279..5298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5532..5549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5564..5703
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5629..5648)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5789..5811
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5805..5996)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
       61 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
      121 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
      181 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
      241 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
      301 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
      361 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg
      421 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
      481 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
      541 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
      601 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
      661 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
      721 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
      781 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
      841 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
      901 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      961 aactgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1021 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     1081 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     1141 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     1201 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1261 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     1321 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1381 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1441 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1501 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1561 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1621 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1681 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1741 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1801 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1861 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1921 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1981 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     2041 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     2101 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     2161 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     2221 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     2281 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     2341 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     2401 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     2461 attttgttta actttaagaa ggagatatac catgcatatg aaaaacatca aaaaaaacca
     2521 ggtaatgaac ctgggtccga actctaaact gctgaaagaa tacaaatccc agctgatcga
     2581 actgaacatc gaacagttcg aagcaggtat cggtctgatc ctgggtgatg cttacatccg
     2641 ttctcgtgat gaaggtaaaa cctactgtat gcagttcgag tggaaaaaca aagcatacat
     2701 ggaccacgta tgtctgctgt acgatcagtg ggtactgtcc ccgccgcaca aaaaagaacg
     2761 tgttaaccac ctgggtaacc tggtaatcac ctggggcgcc cagactttca aacaccaagc
     2821 tttcaacaaa ctggctaacc tgttcatcgt taacaacaaa attaccatcc cgaacaacct
     2881 ggttgaaaac tacctgaccc cgatgtctct ggcatactgg ttcatggatg atggtggtaa
     2941 atgggattac aacaaaaact ctaccaacaa atcgatcgta ctgaacaccc agtctttcac
     3001 tttcgaagaa gtagaatacc tggttaaggg tctgcgtaac aaattccaac tgaactgtta
     3061 cgtaaaaatc aacaaaaaca aaccgatcat ctacatcgat tctatgtctt acctgatctt
     3121 ctacaacctg atcaaaccgt acctgatccc gcagatgatg tacaaactgc cgaacactat
     3181 ctcctccgaa actttcctga aatctcacca ccaccaccac cactaaggat ccggctgcta
     3241 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     3301 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     3361 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
     3421 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
     3481 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
     3541 aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3601 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3661 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3721 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     4021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     4321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4441 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4681 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4921 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5401 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5461 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5521 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5581 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     5641 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5701 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5761 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5821 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5881 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5941 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     6001 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     6061 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     6121 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     6181 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     6241 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     6301 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     6361 cacgttcgct c
//
