LOCUS       sequence-446497-        3415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3415)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 3415)
  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-module5
FEATURES             Location/Qualifiers
     source          1..3415
                     /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        610..685
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(729..746)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(729..745)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(738..760)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(886..1341)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1023..1044)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1235..1254
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1368..1472
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1473..1541,1542..2333)
                     /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(1691..1710)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2504..3092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2993..3012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3246..3263
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3380..3401
                     /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 gcggaacctt gaatgtcgat cgacttgcct tccgcacaat acatcatttc
      181 ttcttagctt tttttcttct tcttcgttca tacagttttt ttttgtttat cagcttacat
      241 tttcttgaac cgtagctttc gttttcttct ttttaacttt ccattcggag tttttgtatc
      301 ttgtttcata gtttgtccca ggattagaat gattaggcat cgaaccttca agaatttgat
      361 tgaataaaac atcttcattc ttaagatatg aagataatct tcaaaaggcc cctgggaatc
      421 tgaaagaaga gaagcaggcc catttatatg ggaaagaaca atagtatttc ttatataggc
      481 ccatttaagt tgaaaacaat cttcaaaagt cccacatcgc ttagataaga aaacgaagct
      541 gagtttatat acagctagag tcgaagtagt gattggggtc ttcaacccag tggacataag
      601 cgaagacccg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      661 tgaaaaagtg gcaccgagtc ggtgcttttt tttttttgtg cgaggaactc tatgtcgaga
      721 ccgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
      781 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
      841 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta
      901 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
      961 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     1021 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     1081 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     1141 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     1201 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     1261 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     1321 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1381 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1441 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1501 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1561 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1621 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1681 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1741 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1801 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1861 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1921 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1981 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2041 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2101 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2161 ctgataaatc tggagccggt gagcgtggtt ctcgcggtat cattgcagca ctggggccag
     2221 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2281 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2341 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2401 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2461 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2521 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2581 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2641 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2701 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2761 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2821 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2881 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2941 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3001 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3061 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3121 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3181 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3241 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3301 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3361 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggc
//
