LOCUS       sequence-392999-        5870 bp    DNA     circular SYN 14-APR-2025
DEFINITION  piggybac vector expressing non-targeting control sgRNA cloned using 
            BlpI and BstXI sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  AUTHORS   Bajpai VK, Swigut T, Mohammed J, Naqvi S, Arreola M, Tycko J, Kim 
            TC, Pritchard JK, Bassik MC, Wysocka J
  TITLE     A genome-wide genetic screen uncovers determinants of human 
            pigmentation.
  JOURNAL   Science. 2023 Aug 11;381(6658):eade6289. doi: 
            10.1126/science.ade6289. Epub 2023 Aug 11.
  PUBMED    37561850
REFERENCE   2  (bases 1 to 5870)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2023-08-11"; volume: "381(6658):eade6289. doi"; pages: " 
            10.1126/science.ade6289. Epub 2023 Aug 11"
COMMENT     Sequence Label: Pb459-mU6-sgRNA-EF1a-PuroR
FEATURES             Location/Qualifiers
     source          1..5870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1482..1516
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(1601..2200)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1684..1704)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2181..2200
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          2216..3154
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2239..2259)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3155..3366)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(3608..3628)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    3632..3665
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   3904..3966
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4096..4113)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4096..4112)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4105..4127)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4254..4709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4341..4360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4551..4572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4735..4839
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4840..4908,4909..5700)
                     /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(5058..5077)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg cccgccgggt
     1021 aactcacggg gtatccatgt ccatttctgc ggcatccagc caggataccc gtcctcgctg
     1081 acgtaatatc ccagcgccgc accgctgtca ttaatctgca caccggcacg gcagttccgg
     1141 ctgtcgccgg tattgttcgg gttgctgatg cgcttcgggc tgaccatccg gaactgtgtc
     1201 cggaaaagcc gcgacgaact ggtatcccag gtggcctgaa cgaacagttc accgttaaag
     1261 gcgtgcatgg ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg ctcaacgtca
     1321 atgcagcagc agtcatcctc ggcaaactct ttccatgccg cttcaacctc gcgggaaaag
     1381 gcacgggctt cttcctcccc gatgcccaga tagcgccagc ttgggcgatg actgagccgg
     1441 aaaaaagacc cgacgatatg atcctgatgc agctagatta accctagaaa gatagtctgc
     1501 gtaaaattga cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg
     1561 tcgctgtgca tttaggacat ctcagtcgcc gctcgaattc ttaggcaccg ggcttgcggg
     1621 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     1681 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     1741 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     1801 ggtcgggcga gacgccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     1861 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     1921 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     1981 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     2041 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccgggtcg acggtgtggc
     2101 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggcccgg gggacgtcgt
     2161 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat gctagctcac gacacctgaa
     2221 atggaagaaa aaaactttga accactgtct gaggcttgag aatgaaccaa gatccaaact
     2281 caaaaagggc aaattccaag gagaattaca tcaagtgcca agctggccta acttcagtct
     2341 ccacccactc agtgtgggga aactccatcg cataaaaccc ctccccccaa cctaaagacg
     2401 acgtactcca aaagcgcgag aactaatcga ggtgcctgga cggcgcccgg tactccgtgg
     2461 agtcacatga agcgacggct gaggacggaa aggccctttt cctttgtgtg ggtgactcac
     2521 ccgcccgctc tcccgagcgc cgcgtcctcc attttgagct ccctgcagca gggccgggaa
     2581 gcggccatct ttccgctcac gcaactggtg ccgaccgggc cagccttgcc gcccagggcg
     2641 gggcgataca cggcggcgcg aggccaggca ccagagcagg ccggccagct tgagactacc
     2701 cccgtccgat tctcggtggc cgcgctcgca ggccccgcct cgccgaacat gtgcgctggg
     2761 acgcacgggc cccgtcgccg cccgcggccc caaaaaccga aataccagtg tgcagatctt
     2821 ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt cgcagcaggt catcaaaaat
     2881 tttaaatggc tagagactta tcgaaagcag cgagacaggc gcgaaggtgc caccagattc
     2941 gcacgcggcg gccccagcgc ccaggccagg cctcaactca agcacgaggc gaaggggctc
     3001 cttaagcgca aggcctcgaa ctctcccacc cacttccaac ccgaagctcg ggatcaagaa
     3061 tcacgtactg cagccaggtg gaagtaattc aaggcacgca agggccataa cccgtaaaga
     3121 ggccaggccc gcgggaacca cacacggcac ttacctgtgt tctggcggca aacccgttgc
     3181 gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa
     3241 aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg
     3301 caccggttca attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct
     3361 ctgcccactg acgggcacat gcatggcggt aatacggtta tccacgcggc cgcctaatgg
     3421 atcctagtac tcgagaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     3481 ctagccttat ttaaacttgc tatgctgttt ccagcttagc tcttaaacta tcgaccctag
     3541 tcggccaaca aggtggttct ccaagggata cttatagtct caaaacacac aattacttta
     3601 cagttagggt gagtttcctt ttgtgctgtt tataacttcg tataatgtat gctatacgaa
     3661 gttatagaaa tccaagccta tcatgtaaaa tgtagctagt attaaaaaga acagattatc
     3721 tgtcttttat cgcacattaa gcctctatag ttactaggaa atattatatg caaattaacc
     3781 ggggcagggg agtagccgag cttctcccac aagtccgtgc gagggggccg gcgcgggcct
     3841 agagatggcg cgtcggatct ctagaattaa taaataaacc tcgatataca gaccgataaa
     3901 acacatgcgt caattttaca catgattatc tttaacgtac gtcacaatat gattatcttt
     3961 ctagggttaa tctagctgcg tgttctgcag cgtgtcgagc atcttcatct gctccatcac
     4021 gctgtaaaac acatttgcac cgcgagtctg cccgtcctcc acgggttcaa aaacgtgaat
     4081 gaacgaggcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4141 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4201 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4261 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4321 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4381 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4441 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4501 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4561 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4621 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4681 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4741 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4801 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4861 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4921 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4981 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5041 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5101 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5161 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5221 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5281 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5341 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5401 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5461 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5521 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5581 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5641 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5701 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5761 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5821 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
