LOCUS       sequence-446496-        3404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            4).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3404)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 3404)
  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-module4
FEATURES             Location/Qualifiers
     source          1..3404
                     /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        611..686
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(718..735)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(718..734)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(727..749)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(875..1330)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1012..1033)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1224..1243
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1357..1461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1462..1530,1531..2322)
                     /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(1680..1699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2493..3081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2982..3001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3235..3252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3369..3390
                     /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 ggcactcatg aggatgtcga tcgacttgcc ttccgcacaa tacatcattt
      181 cttcttagct ttttttcttc ttcttcgttc atacagtttt tttttgttta tcagcttaca
      241 ttttcttgaa ccgtagcttt cgttttcttc tttttaactt tccattcgga gtttttgtat
      301 cttgtttcat agtttgtccc aggattagaa tgattaggca tcgaaccttc aagaatttga
      361 ttgaataaaa catcttcatt cttaagatat gaagataatc ttcaaaaggc ccctgggaat
      421 ctgaaagaag agaagcaggc ccatttatat gggaaagaac aatagtattt cttatatagg
      481 cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg cttagataag aaaacgaagc
      541 tgagtttata tacagctaga gtcgaagtag tgattggggt cttcaaccca gtggacataa
      601 gcgaagaccc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      661 ttgaaaaagt ggcaccgagt cggtgctttt tttttttgtg cggacgagac cgaattcact
      721 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      781 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      841 ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta
      901 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
      961 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     1021 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     1081 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     1141 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     1201 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     1261 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     1321 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1381 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1441 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1501 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1561 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1621 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1681 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     1741 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1801 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1861 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1921 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1981 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2041 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2101 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2161 ggagccggtg agcgtggttc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2221 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2281 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2341 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2401 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2461 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2521 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2581 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     2641 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2701 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2761 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2821 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2881 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2941 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3001 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3061 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3121 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3181 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3241 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     3301 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     3361 aacgcaatta atgtgagtta gctcactcat taggcacccc aggc
//
