LOCUS       sequence-313510-        7553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lenti-viral construct with Cre recombinase and U6 driven sgRNA 
            against mouse Itgb5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7553)
  AUTHORS   Loganathan SK, Schleicher K, Malik A, Quevedo R, Langille E, Teng K,
            Oh RH, Rathod B, Tsai R, Samavarchi-Tehrani P, Pugh TJ, Gingras AC, 
            Schramek D
  TITLE     Rare driver mutations in head and neck squamous cell carcinomas 
            converge on NOTCH signaling.
  JOURNAL   Science. 2020 Mar 13;367(6483):1264-1269. doi: 
            10.1126/science.aax0902.
  PUBMED    32165588
REFERENCE   2  (bases 1 to 7553)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2020-03-13- 13"; volume: "367"; 
            issue: "6483"; pages: "1264-1269"
COMMENT     Sequence Label: pLKO-Cre sgItgb5 v3
FEATURES             Location/Qualifiers
     source          1..7553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        701..927
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             928..1108
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1155..1280
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1773..2006
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2191..2235
                     /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        2533..2781
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2533..2553
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2704..2723
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2803..2878
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2926..3043
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3092..3602
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3521..3539
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      3630..3639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3642..3662
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     LTR             4772..5005
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5077..5198
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5114..5133)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5168..5187
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5238..5373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5393..5412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5394..5412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5422..5439)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5422..5438)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5431..5453)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5580..6035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5667..5686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5877..5898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6061..6165
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6166..6234,6235..7026)
                     /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(6384..6403)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7197..7553,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
      721 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
      781 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
      841 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
      901 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
      961 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     1021 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     1081 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     1141 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     1201 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     1261 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     1321 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     1381 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     1441 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     1501 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     1561 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     1621 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1681 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1741 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1801 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1861 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1921 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     1981 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     2041 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     2101 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     2161 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     2221 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     2281 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     2341 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     2401 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     2461 tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc
     2521 gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2581 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     2641 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2701 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2761 ttgtggaaag gacgaaacac cgaaccgaga tgcccccgag gggttttaga gctagaaata
     2821 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2881 ttttgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2941 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     3001 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     3061 cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca
     3121 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     3181 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     3241 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     3301 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc aggtctcact agtaccctcg
     3361 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     3421 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     3481 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     3541 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     3601 agggggatcc cgggtcgaca tcaagcttgg ccaccatggt gcccaagaag aagaggaaag
     3661 tctccaacct gctgactgtg caccaaaacc tgcctgccct ccctgtggat gccacctctg
     3721 atgaagtcag gaagaacctg atggacatgt tcagggacag gcaggccttc tctgaacaca
     3781 cctggaagat gctcctgtct gtgtgcagat cctgggctgc ctggtgcaag ctgaacaaca
     3841 ggaaatggtt ccctgctgaa cctgaggatg tgagggacta cctcctgtac ctgcaagcca
     3901 gaggcctggc tgtgaagacc atccaacagc acctgggcca gctcaacatg ctgcacagga
     3961 gatctggcct gcctcgccct tctgactcca atgctgtgtc cctggtgatg aggagaatca
     4021 gaaaggagaa tgtggatgct ggggagagag ccaagcaggc cctggccttt gaacgcactg
     4081 actttgacca agtcagatcc ctgatggaga actctgacag atgccaggac atcaggaacc
     4141 tggccttcct gggcattgcc tacaacaccc tgctgcgcat tgccgaaatt gccagaatca
     4201 gagtgaagga catctcccgc accgatggtg ggagaatgct gatccacatt ggcaggacca
     4261 agaccctggt gtccacagct ggtgtggaga aggccctgtc cctgggggtt accaagctgg
     4321 tggagagatg gatctctgtg tctggtgtgg ctgatgaccc caacaactac ctgttctgcc
     4381 gggtcagaaa gaatggtgtg gctgcccctt ctgccacctc ccaactgtcc acccgggccc
     4441 tggaagggat ctttgaggcc acccaccgcc tgatctatgg tgccaaggat gactctgggc
     4501 agagatacct ggcctggtct ggccactctg ccagagtggg tgctgccagg gacatggcca
     4561 gggctggtgt gtccatccct gaaatcatgc aggctggtgg ctggaccaat gtgaacattg
     4621 tgatgaacta catcagaaac ctggactctg agactggggc catggtgagg cttctagagg
     4681 atggggactg aaaatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     4741 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     4801 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     4861 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     4921 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     4981 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     5041 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     5101 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5161 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     5221 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     5281 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     5341 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     5401 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     5461 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     5521 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     5581 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5641 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5701 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5761 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5821 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5881 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5941 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6001 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     6061 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6121 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6181 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6241 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6301 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6361 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6421 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6481 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6541 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6601 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6661 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6721 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6781 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6841 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6901 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6961 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7021 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     7081 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7141 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7201 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7261 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7321 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7381 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7441 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7501 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aac
//
