LOCUS       sequence-436337-        4843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcloning for Golden Gate assembly of CRISPR/Cas9 vectors (module 
            2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4843)
  TITLE     Takeuchi Subcloning Plasmids for Golden Gate assembly of CRISPR/Cas9
            vectors
REFERENCE   2  (bases 1 to 4843)
  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
FEATURES             Location/Qualifiers
     source          1..4843
                     /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"
     promoter        join(606..611,612..629,630..636)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             690..1370
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(756..775)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1690..1995
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1874..1893
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        2044..2119
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2156..2173)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2156..2172)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2165..2187)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2314..2769)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2451..2472)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2663..2682
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2796..2900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2901..2969,2970..3761)
                     /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(3119..3138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3932..4520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4421..4440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4674..4691
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4808..4829
                     /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 cgcggccgca ttaggcaccc
      601 caggctttac actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt
      661 cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg
      721 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
      781 gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa
      841 ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc
      901 cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt
      961 gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg
     1021 acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc
     1081 tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg
     1141 tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt
     1201 tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
     1261 ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
     1321 actgcgatga gtggcaggcg gggcgtaatc tagaggatcc ggcttactaa aagccagata
     1381 acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac
     1441 ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac agttgacagc
     1501 gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa
     1561 ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag
     1621 ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg
     1681 gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
     1741 gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc
     1801 agtgcacgtc tgctgtcaga taaagtcccc cgtgaacttt acccggtggt gcatatcggg
     1861 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
     1921 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
     1981 ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg tcgacgaaga
     2041 cccgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2101 agtggcaccg agtcggtgct tttttttttt tgtgcgaagg ctcgagaccg aattcactgg
     2161 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2221 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2281 cccaacagtt gcgcagcctg aatggcgaat gggaaattgt aaacgttaat attttgttaa
     2341 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     2401 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     2461 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     2521 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     2581 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     2641 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     2701 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     2761 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2821 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2881 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2941 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3001 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3061 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3121 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3181 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3241 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3301 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3361 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3421 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3481 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3541 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3601 gagccggtga gcgtggttct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3661 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3721 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3781 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3841 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3901 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3961 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4021 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4081 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4141 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4201 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4261 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4321 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4381 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4441 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4501 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4561 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4621 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4681 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4741 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4801 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggc
//
