LOCUS       MIGR1_U6gRNA1_PG        7358 bp    DNA     circular SYN 22-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7358)
  AUTHORS   City of Hope
  TITLE     Direct Submission
  JOURNAL   Exported Mar 28, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             123..639
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    703..1044
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1111..1527
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1457..1479
                     /label=LXSN F
     primer_bind     1540..1576
                     /label=U6g1 F2
                     /note="for U6gRNA1 v3"
     promoter        1558..1798
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    1815..2431
                     /label=filler
     misc_RNA        2440..2515
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2499..2531)
                     /label=U6g2 R4
     misc_feature    complement(2502..2522)
                     /label=gib_sgRNAlib_rev_scaffold
     primer_bind     2522..2561
                     /label=PGK F2
     misc_feature    complement(2523..2543)
                     /label=gib_sgRNAlib_rev_overlapping
     misc_feature    2538..3037
                     /label=PGK promoter
     primer_bind     complement(2566..2586)
                     /label=PGK-N R
     regulatory      3056..3061
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3062..3763
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(3744..3806)
                     /label=TagBFP R1
     misc_feature    3813..3818
                     /label=remove BspMI
     LTR             3875..4389
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4551..4567
                     /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)."
     promoter        complement(join(4575..4581,4582..4599,4600..4605))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4620..4641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4929..5517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5688..6479,6480..6548))
                     /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"
     promoter        complement(6549..6653)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag
       61 cctatagagt acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg
      121 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
      181 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac
      241 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      301 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat
      361 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
      421 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
      481 acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc
      541 aataaagcct cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc
      601 ctcagattga ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg
      661 gagacccctg cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt
      721 ttcgtgtctg tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt
      781 ctgtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc
      841 tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc
      901 ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta
      961 ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac
     1021 cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg
     1081 actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg
     1141 accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag
     1201 aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg
     1261 cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct
     1321 ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt
     1381 gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca
     1441 tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat
     1501 ccagccctca ctccttctct aggcgccgga attagatctc tcgaggttaa cgaattcgag
     1561 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1621 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1681 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1741 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1801 aacaccgggt cttcgcaatt tgagttaagg gtgttgctct caatgatttc attaatggtt
     1861 caatttttaa tttcttttct tctggtttaa aattcaagtt taaagtgaaa gtgtaatatg
     1921 cacccatttc tttaaataaa tcttctaaat agtctactaa tgttttattt tgttttttat
     1981 aaaatcaagc agcctctgct attaatatag aagcttgtat tccatcttta tctctagctg
     2041 agtcatcaat tacatatcca taactttctt cataagcaaa aacaaaattt aatccgttat
     2101 cttcttcttt agcaatttct ctacccattc atttaaatcc agttaaagtt tttacaatat
     2161 taactccata tttttcatga gcgattctat cacccaaatc acttgttaca aaacttgaat
     2221 atagagccgg attttttgga atgctattta agcgttttag atttgataat tttcaatcaa
     2281 ttaaaattgg tcctgtttga tttccatcta atcttacaaa atgaccatca tgttttattg
     2341 ccattccaaa tctgtcagca tctgggtcat tcataataat aatatctgca tcatgtttaa
     2401 taccatattc aagcggtatt tttcatgcag ggaagaccag ttttagagct agaaatagca
     2461 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2521 ttggatccaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     2581 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     2641 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     2701 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     2761 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     2821 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     2881 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     2941 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     3001 cgcgctgttc tcctcttcct catctccggg cctttcggac cggttctaga gcgctgccac
     3061 catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     3121 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     3181 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     3241 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     3301 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg
     3361 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     3421 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     3481 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     3541 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     3601 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     3661 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     3721 atactgcgac ctccctagca aactggggca caagcttaat tgagcggccg ctgaattaat
     3781 taagaattat caagcttatc gataccgtcg actcctgcag ccaagcttat cgataaaata
     3841 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     3901 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag
     3961 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg
     4021 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc
     4081 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     4141 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     4201 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     4261 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     4321 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     4381 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga
     4441 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct
     4501 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg
     4561 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4621 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4681 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4741 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4801 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4861 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4921 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4981 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5041 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5101 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5161 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5221 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5281 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5341 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5401 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5461 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5521 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5581 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5641 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5701 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5761 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5821 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5881 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5941 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6001 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6061 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6121 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6181 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6241 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6301 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6361 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6421 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6481 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6541 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6601 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6661 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6721 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     6781 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     6841 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     6901 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     6961 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     7021 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     7081 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg
     7141 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     7201 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     7261 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     7321 cgatgcgtcc ggcgtagagg cgattagtcc aatttgtt
//
