LOCUS       sequence-275065-        8486 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SIX1 CRISPRi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8486)
  TITLE     Kingsbury Lab Plasmids
REFERENCE   2  (bases 1 to 8486)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pTJK475
FEATURES             Location/Qualifiers
     source          1..8486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        complement(2101..2349)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2159..2178)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2329..2349)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2380..2496
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2700..3882
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2931..3873
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3830..3850
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4511..5099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4564..4584)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4982..4993)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5186..5419
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5491..5612
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5528..5547)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5582..5601
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5652..5787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5807..5826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5808..5826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5836..5853)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5836..5852)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5845..5867)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5994..6449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6081..6100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6291..6312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6475..6579
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6580..6648,6649..7440)
                     /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(6798..6817)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7611..8199
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8100..8119
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8353..8370
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     1921 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcggtta
     1981 acactagaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     2041 atttaaactt gctatgctgt ttccagcata gctcttaaac cggcaactag cagcatccac
     2101 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     2161 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     2221 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     2281 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     2341 taggccctct tcctgcccga ccttggtacc ggatccaact tttaaaagaa aaggggggat
     2401 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2461 agaattacaa aaacaaatta caaaattcaa aattttatcg atgcctcccc gtcaccaccc
     2521 cccccaaccc gccccgaccg gagctgagag taattcatac aaaaggactc gcccctgcct
     2581 tggggaatcc cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg
     2641 ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag
     2701 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2761 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2821 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2881 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     2941 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     3001 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     3061 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     3121 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     3181 gctttcgata agtctctagc catttaaaat ttttgatgat atcctgcgac gctttttttt
     3241 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     3301 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     3361 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     3421 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     3481 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     3541 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     3601 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     3661 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     3721 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     3781 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     3841 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaaaactacc cctctagtac
     3901 cgccatgacc gagtacaagc ctactgttcg gctcgcgact cgcgatgatg tccccagagc
     3961 agtgagaaca ctggctgctg cctttgccga ttaccccgca acccgccata ccgtggatcc
     4021 cgaccggcac atcgagaggg tgactgaact gcaggaattg tttttgacaa gggtgggact
     4081 cgacattgga aaggtgtggg tggccgatga cggagccgcc gtcgccgttt ggaccacacc
     4141 agagagcgtc gaagccggcg cggtgttcgc cgagatcggc ccaagaatgg ccgagctctc
     4201 tggatctaga ctggccgcgc aacagcagat ggaaggcctg ctcgcgccac accgacctaa
     4261 ggagcccgca tggttcctgg ccaccgtagg cgtttccccc gatcaccagg gaaaggggct
     4321 tggcagcgcc gtagttctgc ccggcgtgga ggccgctgaa cgagctggtg ttcccgcatt
     4381 tcttgagacc agtgcaccta ggaaccttcc tttctatgaa aggttgggtt tcaccgtcac
     4441 cgccgatgtg gaagtgcctg agggacctcg aacctggtgc atgactcgaa agcctggcgc
     4501 ctaactcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     4561 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     4621 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     4681 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     4741 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     4801 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     4861 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
     4921 atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     4981 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     5041 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
     5101 tggaattcga gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     5161 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     5221 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5281 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5341 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5401 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     5461 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     5521 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5581 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     5641 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5701 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     5761 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     5821 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     5881 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     5941 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     6001 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     6061 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6121 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6181 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     6241 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6301 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     6361 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6421 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     6481 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6541 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6601 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6661 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6721 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6781 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6841 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6901 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6961 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7021 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7081 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7141 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7201 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7261 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     7321 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7381 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7441 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7501 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7561 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7621 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7681 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7741 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7801 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7861 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7921 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7981 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     8041 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     8101 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8161 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8221 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     8281 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     8341 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     8401 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     8461 aaagcgggca gtgagcgcaa cgcaat
//
