LOCUS       sequence-383680-        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 K223I 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) K223I
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2174..2193
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2213..2290)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2496..2515
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2599..2618
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2599..2617
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2618..2642
                     /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             2657..2679
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3400..3417
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3471..3489)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3485..3532
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3707..3712,3713..3729,3730..3735))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3762..3780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3848..3952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3953..4021,4022..4813)
                     /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(4171..4190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4984..5572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5473..5492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5726..5743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5758..5897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5823..5842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5983..6005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5999..6190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
       61 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
      121 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
      181 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
      241 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
      301 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
      361 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
      421 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
      481 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
      541 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
      601 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
      661 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
      721 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
      781 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
      841 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
      901 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
      961 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     1021 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     1081 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     1141 ctttccagtc gggaaactgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1201 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1261 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1321 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1381 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1441 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1501 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1561 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1621 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1681 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1741 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1801 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1861 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1921 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1981 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2041 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     2101 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     2161 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     2221 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     2281 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     2341 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     2401 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     2461 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     2521 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     2581 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     2641 cccctctaga aataattttg tttaacttta agaaggagat ataccatgca tatgaaaaac
     2701 atcaaaaaaa accaggtaat gaacctgggt ccgaactcta aactgctgaa agaatacaaa
     2761 tcccagctga tcgaactgaa catcgaacag ttcgaagcag gtatcggtct gatcctgggt
     2821 gatgcttaca tccgttctcg tgatgaaggt aaaacctact gtatgcagtt cgagtggaaa
     2881 aacaaagcat acatggacca cgtatgtctg ctgtacgatc agtgggtact gtccccgccg
     2941 cacaaaaaag aacgtgttaa ccacctgggt aacctggtaa tcacctgggg cgcccagact
     3001 ttcaaacacc aagctttcaa caaactggct aacctgttca tcgttaacaa caaaaaaacc
     3061 atcccgaaca acctggttga aaactacctg accccgatgt ctctggcata ctggttcatg
     3121 gatgatggtg gtaaatggga ttacaacaaa aactctacca acaaatcgat cgtactgaac
     3181 acccagtctt tcactttcga agaagtagaa tacctggtta agggtctgcg taacaaattc
     3241 caactgaact gttacgtaaa aatcaacaaa aacaaaccga tcatctacat cgattctatg
     3301 tcttacctga tcttctacaa cctgatcaaa ccgtacctca tcccccagat gatgtacatt
     3361 ctgccgaaca ctatctcctc cgaaactttc ctgaaatctc accaccacca ccaccactaa
     3421 ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3481 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3541 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     3601 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     3661 cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg
     3721 atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt
     3781 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3841 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3901 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3961 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4021 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4081 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4141 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     4201 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4261 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4321 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4381 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4441 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     4501 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4561 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4621 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4681 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4741 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4801 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4861 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4921 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4981 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5041 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5101 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     5161 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5221 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5281 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5341 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5401 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5461 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5521 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5581 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5641 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5701 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     5761 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     5821 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     5881 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5941 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6001 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     6061 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     6121 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     6181 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     6241 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     6301 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     6361 agggtcaatg c
//
