LOCUS       sequence-446499-        3302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3302)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 3302)
  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-module6
FEATURES             Location/Qualifiers
     source          1..3302
                     /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"
     primer_bind     complement(616..633)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(616..632)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(625..647)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(773..1228)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(910..931)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1122..1141
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1255..1359
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1360..1428,1429..2220)
                     /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(1578..1597)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2391..2979
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2880..2899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3133..3150
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3267..3288
                     /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 gatgtcgatc gacttgcctt ccgcacaata catcatttct tcttagcttt
      181 ttttcttctt cttcgttcat acagtttttt tttgtttatc agcttacatt ttcttgaacc
      241 gtagctttcg ttttcttctt tttaactttc cattcggagt ttttgtatct tgtttcatag
      301 tttgtcccag gattagaatg attaggcatc gaaccttcaa gaatttgatt gaataaaaca
      361 tcttcattct taagatatga agataatctt caaaaggccc ctgggaatct gaaagaagag
      421 aagcaggccc atttatatgg gaaagaacaa tagtatttct tatataggcc catttaagtt
      481 gaaaacaatc ttcaaaagtc ccacatcgct tagataagaa aacgaagctg agtttatata
      541 cagctagagt cgaagtagtg attggggtct tcaacccagt ggacataagc gaagacccgt
      601 ttcgagaccg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
      661 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
      721 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
      781 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
      841 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
      901 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
      961 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1021 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1081 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1141 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1201 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1261 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1321 ccctgataaa tgcttcaata atattgaaaa gggaagagta tgagtattca acatttccgt
     1381 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1441 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1501 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1561 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1621 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1681 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1741 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1801 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1861 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     1921 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1981 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2041 tttattgctg ataaatctgg agccggtgag cgtggttctc gcggtatcat tgcagcactg
     2101 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2161 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2221 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2281 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2341 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2401 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2461 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2521 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     2581 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2641 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2701 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2761 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2821 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2881 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2941 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3001 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3061 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3121 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     3181 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3241 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     3301 gc
//
