LOCUS       sequence-446490-        2966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2966)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 2966)
  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"
COMMENT     Sequence Label: pT7-module1
FEATURES             Location/Qualifiers
     source          1..2966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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)."
     primer_bind     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     98..117
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        98..116
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        170..245
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(280..297)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(280..296)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(289..311)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(437..892)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(574..595)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     786..805
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        919..1023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1024..1092,1093..1884)
                     /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     complement(1242..1261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2055..2643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2544..2563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2797..2814
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2931..2952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagctctaa tacgactcac tatagggaaa
      121 gcttggtctc aattggggtc ttcaacccag tggacataag cgaagacccg ttttagagct
      181 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      241 ggtgcttttt tttttttgtg cgctcgcgag accgaattca ctggccgtcg ttttacaacg
      301 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
      361 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
      421 cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
      481 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
      541 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
      601 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
      661 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
      721 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
      781 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
      841 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
      901 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      961 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1021 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1081 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1141 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1201 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1261 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1321 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1381 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1441 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     1501 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     1561 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     1621 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     1681 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt
     1741 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     1801 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     1861 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1921 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2161 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     2221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2761 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2821 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2881 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     2941 tagctcactc attaggcacc ccaggc
//
