LOCUS       MIGR1-U6gRNA2-PG        8336 bp    DNA     circular SYN 04-MAR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8336)
  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..8336
                     /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     1518..1564
                     /label=U6g1 F
     promoter        1546..1786
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1546..1564
                     /label=U6g4 F
     primer_bind     1785..1821
                     /label=Filler2 F2
     primer_bind     1789..1821
                     /label=Filler2 F3
     misc_feature    1803..2419
                     /label=filler
     primer_bind     complement(2398..2419)
                     /label=Filler2 R2
     primer_bind     complement(2398..2419)
                     /label=Filler2 R3
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2487..2511)
                     /label=U6g2 R4
     primer_bind     complement(2493..2534)
                     /label=U6g1 R4
     primer_bind     2525..2550
                     /label=U6g4 F
     promoter        2532..2772
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2532..2550
                     /label=U6g1 F
     primer_bind     2771..2809
                     /label=Filler2 F2
     primer_bind     2775..2809
                     /label=Filler2 F3
     misc_feature    2791..3407
                     /label=filler
     primer_bind     complement(3386..3421)
                     /label=Filler2 R2
     primer_bind     complement(3386..3421)
                     /label=Filler2 R3
     misc_RNA        3418..3493
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3477..3509)
                     /label=U6g2 R4
     misc_feature    complement(3480..3500)
                     /label=gib_sgRNAlib_rev_scaffold
     primer_bind     complement(3483..3501)
                     /label=U6g1 R4
     primer_bind     3500..3539
                     /label=PGK F2
     misc_feature    complement(3501..3521)
                     /label=gib_sgRNAlib_rev_overlapping
     misc_feature    3516..4015
                     /label=PGK promoter
     primer_bind     complement(3994..4042)
                     /label=PGK R
     regulatory      4034..4039
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4040..4741
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(4161..4181)
                     /label=seq BFP R
     primer_bind     complement(4722..4784)
                     /label=TagBFP R1
     LTR             4853..5367
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5529..5545
                     /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(5553..5559,5560..5577,5578..5583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5598..5619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5907..6495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6666..7457,7458..7526))
                     /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(7527..7631)
                     /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 tcgaggaggg cctatttccc
     1561 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     1621 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1681 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1741 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct
     1801 tcgcaatttg agttaagggt gttgctctca atgatttcat taatggttca atttttaatt
     1861 tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca cccatttctt
     1921 taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa aatcaagcag
     1981 cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag tcatcaatta
     2041 catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct tcttctttag
     2101 caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta actccatatt
     2161 tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat agagccggat
     2221 tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt aaaattggtc
     2281 ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc attccaaatc
     2341 tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata ccatattcaa
     2401 gcggtatttt tcatgcaggg aagaccagtt ttagagctag aaatagcaag ttaaaataag
     2461 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gcacagcgcg
     2521 tatgtgaatt cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2581 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2641 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2701 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2761 tgtggaaagg acgaaacacc ggaggcaggt gcaatttgag ttaagggtgt tgctctcaat
     2821 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     2881 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     2941 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     3001 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     3061 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     3121 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     3181 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     3241 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     3301 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     3361 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggacc tgcctcagtt
     3421 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3481 accgagtcgg tgcttttttt ggatccaatt ctaccgggta ggggaggcgc ttttcccaag
     3541 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     3601 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     3661 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     3721 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga
     3781 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     3841 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     3901 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     3961 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcggaccg
     4021 gttctagagc gctgccacca tgagcgagct gattaaggag aacatgcaca tgaagctgta
     4081 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
     4141 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     4201 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
     4261 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
     4321 cacatacgag gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     4381 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
     4441 gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct
     4501 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
     4561 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
     4621 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     4681 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaattg
     4741 agcggccgct gaattaatta agaattatca agcttatcga taccgtcgac tcctgcagcc
     4801 aagcttatcg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     4861 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat
     4921 acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg
     4981 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5041 gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     5101 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     5161 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     5221 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc
     5281 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     5341 gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat
     5401 tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc
     5461 acatactcca atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa
     5521 ttgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5581 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5641 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5701 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5761 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5821 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5881 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5941 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6001 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6061 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6121 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6181 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6241 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6301 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6361 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6421 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6481 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6541 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6601 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6661 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6721 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6781 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6841 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6901 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6961 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7021 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7081 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7141 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7201 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7261 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7321 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7381 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7441 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7501 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7561 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7621 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7681 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     7741 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     7801 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     7861 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     7921 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     7981 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     8041 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     8101 cgttgtaaaa cgacggcgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     8161 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     8221 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     8281 cgccggtgat gccggccacg atgcgtccgg cgtagaggcg attagtccaa tttgtt
//
