LOCUS       sequence-136960-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBT264-U6p-{2xBbsI}-sgRNA scaffold-{MfeI}.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4374)
  AUTHORS   Wong AS, Choi GC, Cui CH, Pregernig G, Milani P, Adam M, Perli SD, 
            Kazer SW, Gaillard A, Hermann M, Shalek AK, Fraenkel E, Lu TK
  TITLE     Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Feb 10. pii: 201517883.
  PUBMED    26864203
REFERENCE   2  (bases 1 to 4374)
  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: "Proc Natl 
            Acad Sci U S A. 2016 Feb 10. pii: 201517883."
COMMENT     Sequence Label: pAWp28
FEATURES             Location/Qualifiers
     source          1..4374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2714..2962
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2714..2734
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2885..2904
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2997..3072
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    3119..3200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3156..3175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3730..3749)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3731..3749)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3756..3773)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3756..3772)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3765..3787)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3914..4369
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4001..4020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4211..4232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctgatttaa atggggacag
     2221 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     2281 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     2341 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     2401 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgagtgc
     2461 acggggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
     2521 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     2581 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     2641 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta
     2701 ggctgaagat ctagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2761 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2821 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2881 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2941 cttgtggaaa ggacgaaaca ccgggtcttc gctgcactcg agtgcagcga agacctgttt
     3001 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     3061 ccgagtcggt gcttttttgg atccgcaacg gaattcctga cgcacaattg ttgttgttaa
     3121 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3181 taaagcattt ttttcactgc ctcgaatcac tagtggggac agcccccccc caaagccccc
     3241 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     3301 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     3361 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     3421 gacacctggg gggatacggg gaaaaagctt taggctgagt gcacggggac agcccccccc
     3481 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
     3541 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
     3601 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
     3661 tgagcctgca gacacctggg gggatacggg gaaaaagctt taggctgagg cgcgcctgta
     3721 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     3781 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3841 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3901 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3961 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4021 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4081 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4141 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4201 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4261 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4321 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtg
//
