LOCUS       sequence-274132-        3988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for making sgRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3988)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 3988)
  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"; journalName: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: pT7gRNA-ccdb
FEATURES             Location/Qualifiers
     source          1..3988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      171..759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     660..679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     913..930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1047..1068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1083..1088,1089..1106,1107..1113)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1121..1137
                     /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     1126..1148
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1145..1161
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1145..1161
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1185..1204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1185..1203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(1256..1561)
                     /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     complement(1358..1377)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1903..2562)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2477..2496
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2616..2622,2623..2640,2641..2646))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_RNA        2684..2759
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2787..2804)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2787..2803)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2796..2818)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        3023..3127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3128..3196,3197..3988)
                     /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(3346..3365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
       61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
      361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      541 ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg
      601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      781 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
      841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
      901 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
      961 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1021 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     1081 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     1141 cacacaggaa acagctatga ccatgattac gccaagcttg aaattaatac gactcactat
     1201 aggggagacg agatcttcga cctgcagact ggctgtgtat aagggagcct gacatttata
     1261 ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag
     1321 ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc
     1381 agctttcatc cccgatatgc accaccgggt aaagttcacg ggggacttta tctgacagca
     1441 gacgtgcact ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct
     1501 gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca
     1561 tttcaccagc ccctgttctc gtcggcaaag gagccgttca tttcaataaa ccgggcgacc
     1621 tcagccatcc cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca
     1681 ttctgcatgg ttgtgcttac cgaaccggag atattgacat catatatgcc ttgagcaact
     1741 gatagctgtc gctgtcaact gtcactgtaa tacgctgctt catagcatac ctctttttga
     1801 catacttcgg gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata
     1861 cgcatactgt tatctggctt ttagtaagcc ggatcctcta gattacgccc cgccctgcca
     1921 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     1981 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     2041 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     2101 ggtgaaactc acccagggat tggctgaaac gaaaaacata ttctcaataa accctttagg
     2161 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     2221 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     2281 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     2341 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     2401 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     2461 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     2521 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     2581 tgaaaatctc gacggatcct aactcaaaat ccacacatta tacgagccgg aagcataaag
     2641 tgtaaagcct gggggtgcct aatgcggccg gtcgaccgtc tctgttttag agctagaaat
     2701 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg
     2761 gatccccggg taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa
     2821 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2881 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2941 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataagg
     3001 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3061 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3121 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3181 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3241 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3301 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3361 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3421 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3481 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3541 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3601 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3661 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3721 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3781 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3841 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3901 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3961 aggtgcctca ctgattaagc attggtaa
//
