LOCUS       sequence-325147-        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      202..790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     691..710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     944..961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1078..1099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1114..1119,1120..1137,1138..1144)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1152..1168
                     /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     1157..1179
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1176..1192
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1176..1192
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1216..1235
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1216..1234
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(1287..1592)
                     /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(1389..1408)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1934..2593)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2508..2527
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2647..2653,2654..2671,2672..2677))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_RNA        2715..2790
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2818..2835)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2818..2834)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2827..2849)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        3054..3158
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3159..3227,3228..3988,1..31)
                     /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(3377..3396)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
       61 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
      121 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
      181 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      241 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      301 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      361 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      421 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      481 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      541 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      601 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      661 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      721 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      781 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      841 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      901 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      961 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1021 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     1081 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     1141 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     1201 cgccaagctt gaaattaata cgactcacta taggggagac gagatcttcg acctgcagac
     1261 tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt aatggcgttt
     1321 ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc
     1381 acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg
     1441 taaagttcac gggggacttt atctgacagc agacgtgcac tggccagggg gatcaccatc
     1501 cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc
     1561 tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct cgtcggcaaa
     1621 ggagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat tttccgcttt
     1681 ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta ccgaaccgga
     1741 gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac tgtcactgta
     1801 atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata tcagtatata
     1861 ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct tttagtaagc
     1921 cggatcctct agattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa
     1981 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     2041 tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt
     2101 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaaa
     2161 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     2221 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     2281 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     2341 atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc
     2401 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     2461 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     2521 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     2581 tttttttctc cattttagct tccttagctc ctgaaaatct cgacggatcc taactcaaaa
     2641 tccacacatt atacgagccg gaagcataaa gtgtaaagcc tgggggtgcc taatgcggcc
     2701 ggtcgaccgt ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     2761 caacttgaaa aagtggcacc gagtcggtgc ggatccccgg gtaccgagct cgaattcact
     2821 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2881 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2941 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     3001 gcatctgtgc ggtatttcac accgcataag gtggcacttt tcggggaaat gtgcgcggaa
     3061 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3121 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3181 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3241 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3301 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3361 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3421 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3481 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3541 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3601 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3661 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3721 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3781 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3841 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3901 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3961 tggatgaacg aaatagacag atcgctga
//
