LOCUS       sequence-258959-        2553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector useful for golden gate or other seamless cloning 
            technique based on Type IIS restriction enzymes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2553)
  AUTHORS   Altaib H, Ozaki Y, Kozakai T, Badr Y, Nomura I, Suzuki T
  TITLE     A New Escherichia coli Entry Vector Series (pIIS18) for Seamless 
            Gene Cloning Using Type IIS Restriction Enzymes.
  JOURNAL   Microbiol Resour Announc. 2019 Oct 10;8(41). pii: 8/41/e00323-19. 
            doi: 10.1128/MRA.00323-19.
  PUBMED    31601653
REFERENCE   2  (bases 1 to 2553)
  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: "Microbiol 
            Resour Announc."; date: "2019-10-10"; volume: "8(41). pii: 
            8/41/e00323-19. doi"; pages: " 10.1128/MRA.00323-19"
COMMENT     Sequence Label: pIIS18-BsaI
FEATURES             Location/Qualifiers
     source          1..2553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(61..387)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITNSSSVPGDPVSRDRCRHASLALAVVLQRRDWENPGVTQLN
                     RLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTIC
                     SDAA"
     primer_bind     66..85
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     279..301
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     293..310
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     294..310
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(383..399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(383..399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(396..418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    407..423
                     /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)."
     promoter        complement(join(431..437,438..455,456..461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    476..497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(614..631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(785..1373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(865..884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1544..2335,2336..2404))
                     /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     2167..2186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2405..2509)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
       61 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
      121 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
      181 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      241 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      301 gacggccagt gccaagcttg catgcctgca gcggtctcgc gagaccggat ccccgggtac
      361 cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      421 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      481 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      541 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      601 gctattccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      661 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      721 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      781 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      841 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      901 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      961 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1021 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1081 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1141 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1201 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1261 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1321 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1381 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1441 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1501 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     1561 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1621 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1681 cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1741 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1801 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1861 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1921 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1981 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2041 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     2101 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     2161 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     2221 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     2281 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     2341 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     2401 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     2461 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     2521 gaaaagtgcc acctgacgtc taagaaacca tta
//
