LOCUS       sequence-446504-        3413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            2*5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3413)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 3413)
  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-module2*5
FEATURES             Location/Qualifiers
     source          1..3413
                     /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        608..683
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(727..744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(727..743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(736..758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(884..1339)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1021..1042)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1233..1252
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1366..1470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1471..1539,1540..2331)
                     /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(1689..1708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2502..3090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2991..3010
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3244..3261
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3378..3399
                     /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 gctcgatcga atgtcgatcg acttgccttc cgcacaatac atcatttctt
      181 cttagctttt tttcttcttc ttcgttcata cagttttttt ttgtttatca gcttacattt
      241 tcttgaaccg tagctttcgt tttcttcttt ttaactttcc attcggagtt tttgtatctt
      301 gtttcatagt ttgtcccagg attagaatga ttaggcatcg aaccttcaag aatttgattg
      361 aataaaacat cttcattctt aagatatgaa gataatcttc aaaaggcccc tgggaatctg
      421 aaagaagaga agcaggccca tttatatggg aaagaacaat agtatttctt atataggccc
      481 atttaagttg aaaacaatct tcaaaagtcc cacatcgctt agataagaaa acgaagctga
      541 gtttatatac agctagagtc gaagtagtga ttggggtctt caacccagtg gacataagcg
      601 aagacccgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      661 aaaaagtggc accgagtcgg tgcttttttt tttttgtgcg aggaactcta tgtcgagacc
      721 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
      781 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
      841 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat
      901 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
      961 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     1021 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     1081 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     1141 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     1201 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     1261 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     1321 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1441 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1501 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1561 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1621 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1681 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1741 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1801 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1861 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1921 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1981 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     2041 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2101 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2161 gataaatctg gagccggtga gcgtggttct cgcggtatca ttgcagcact ggggccagat
     2221 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2281 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2341 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2401 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2461 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2521 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2581 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2641 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2701 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2761 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2821 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2881 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2941 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3001 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3061 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3121 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3181 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3241 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     3301 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     3361 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggc
//
