LOCUS       sequence-446494-        3399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3399)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 3399)
  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-module3
FEATURES             Location/Qualifiers
     source          1..3399
                     /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"
     misc_RNA        606..681
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(713..730)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(713..729)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(722..744)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(870..1325)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1007..1028)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1219..1238
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1352..1456
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1457..1525,1526..2317)
                     /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(1675..1694)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2488..3076
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2977..2996
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3230..3247
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3364..3385
                     /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 catagggaaa
      121 gcttggtctc gggcttcaat gtcgatcgac ttgccttccg cacaatacat catttcttct
      181 tagctttttt tcttcttctt cgttcataca gttttttttt gtttatcagc ttacattttc
      241 ttgaaccgta gctttcgttt tcttcttttt aactttccat tcggagtttt tgtatcttgt
      301 ttcatagttt gtcccaggat tagaatgatt aggcatcgaa ccttcaagaa tttgattgaa
      361 taaaacatct tcattcttaa gatatgaaga taatcttcaa aaggcccctg ggaatctgaa
      421 agaagagaag caggcccatt tatatgggaa agaacaatag tatttcttat ataggcccat
      481 ttaagttgaa aacaatcttc aaaagtccca catcgcttag ataagaaaac gaagctgagt
      541 ttatatacag ctagagtcga agtagtgatt ggggtcttca acccagtgga cataagcgaa
      601 gacccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      661 aaagtggcac cgagtcggtg cttttttttt tttgtgcacc gagaccgaat tcactggccg
      721 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
      781 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
      841 aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt
      901 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
      961 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     1021 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     1081 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     1141 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     1201 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     1261 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     1321 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1381 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1441 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1501 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1561 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1621 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1681 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1741 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1801 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1861 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1921 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1981 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2041 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2101 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2161 cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2221 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2281 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2341 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2401 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2461 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2521 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2581 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2641 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2701 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2761 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2821 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2881 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2941 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3001 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3061 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3121 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3181 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3241 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3301 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3361 aattaatgtg agttagctca ctcattaggc accccaggc
//
