LOCUS       sequence-158533-        6848 bp    DNA     circular SYN 15-APR-2025
DEFINITION  retroviral expression of random shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6848)
  TITLE     Rothenberg lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6848)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Banshee shRandom mCherry
FEATURES             Location/Qualifiers
     source          1..6848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..91
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    208..229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(244..249,250..267,268..274)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    282..298
                     /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     287..309
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     306..322
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     306..322
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        343..361
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     398..414
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     398..414
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(1375..1623)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1433..1452)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1603..1623)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1967..1986)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(2092..2802)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2370..2389)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2639..2657
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2799..2808
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2854..3270)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(2870..2886)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(2902..2924)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    3337..3678
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             3742..3917
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     primer_bind     complement(4379..4398)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4380..4398)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4405..4422)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4405..4421)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4414..4436)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4563..5018
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4650..4669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4860..4881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5044..5148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5149..5217,5218..6009)
                     /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(5367..5386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6180..6768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6669..6688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
       61 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
      121 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      181 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      241 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      301 tcacacagga aacagctatg accatgatta cgccaagctc gaaattaacc ctcactaaag
      361 ggaacaaaag ctggagctcc accgcggtgg cggccgctct agaactagtg gatctgtctg
      421 tacacagggg caggacccct ttttatggca aaatctcaca ctgagcccag gcaacctttt
      481 cacagaagcc tgccctcaga gtattaacta ctctgcaatc gccgtgctct caggaaggga
      541 gtgcaaacta gaaatttaaa atacggagaa gccagcaaaa tagacgagga tagattgact
      601 aaatccctca tccaggaggc ataaaaatct taaaagtcgt gtggcagata gtccacagag
      661 cccttcaaca ttcttttata aagccgagag tcaggatgta gtctggcttg aagggaacac
      721 tgttcttgca ttttattata aacaaaaacc gggctgacgg agtccctgcg gcacatcagg
      781 aatgttagtg agacctgtgt aggtgggaga aactgaaaga cccccgctga cgggtagtca
      841 atcactcaga ggagaccctc ccaaggaaca gcgagaccac gagtcggatg caactgcaag
      901 agggtttatt gagaacacgg gtacccgggc gacgcagtct atcggaggac tggcgcgccg
      961 agtgaggggt tgtgggctct tttattgagc tcggggagca gaagcgcgcg aacagaagcg
     1021 agaagcgaac tgattggtta gttcaaataa ggcacagggt catttcaggt ccttggggca
     1081 ccctggaaac atctgatggt tctctagaaa ctgctgaggg cgggaccgca tctggggacc
     1141 atctgttctt ggccctgagc cggggcagga actgcttacc acagatatcc tgtttggccc
     1201 atattctgct gttccaactg ttcttggccc tgagccgggg caggaactgc ttaccacaga
     1261 tatcctgttt ggcccatatt ctgctgtctc tctgtttctg accttgatcc aagcttaaaa
     1321 aacctaaggt taagtcgccc tcgtctcttg aacgagggcg acctaaccct aggcggtgtt
     1381 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
     1441 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
     1501 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
     1561 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
     1621 ctcttcctgc ccgaccttgg atccagatct ttcattcccc cctttttctg gagactaaat
     1681 aaaatctttt attctatcta tggctcgtac tctataggct tcagctgtga tattgttgag
     1741 tcaaaactag agcctggacc actgatatcc tgtctttaac aaattggact aatcgatacc
     1801 gtcgactcga gacgcgtcta gaggatccgt cgactaccga accccagagt cccgctcaga
     1861 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     1921 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     1981 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     2041 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg cttacttgta
     2101 cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat
     2161 ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg
     2221 cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc
     2281 gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat
     2341 ccgctcggag gaggcctccc agcccattgt cttcttctgc attacggggc cgtcggaggg
     2401 gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc
     2461 ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
     2521 ctcggggaag gacagcttca agtagtcggg aatgtcggcg gggtgcttca cgtaggcctt
     2581 ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt
     2641 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
     2701 gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc
     2761 cttgatgatg gccatgttat cctcctcgcc cttgctcacc atggtggcga ccggtggatc
     2821 ccgggcccgc ggtaccgtcg actgcagaat tccggcgcct agagaaggag tgagggctgg
     2881 ataaagggag gatcgaggcg gggtcgaacg aggaggttca agggggagag acggggcgga
     2941 tggaggaaga ggaggcggag gcttagggtg tacaaagggc ttgacccagg gaggggggtc
     3001 aaaagccaag gcttcccagg tcacgatgta ggggacctgg tctgggtgtc catgcgggcc
     3061 aggtgaaaag accttgatct taacctgggt gatgaggtct cggttaaagg tgccgtctcg
     3121 cggccatccg acgttaaagg ttggccattc tgcagagcag aaggtaaccc aacgtctctt
     3181 cttgacatct accgactggt tgtgagcgat ccgctcgaca tctttccagt gacctaaggt
     3241 caaacttaag ggagtggtaa cagtctggcc ctaattttca gacaaataca gaaacacagt
     3301 cagacagaga caacacagaa cgatgctgca gcgctgcagc agacaagacg cgcggcttcg
     3361 gttccaaacc gaaagcaaaa attcagacgg aggcgggaac tgttttaggt tctcgtctcc
     3421 taccagaacc acatatcctg acggggtcgg attccacatc gactcccttc ctcaggtcgg
     3481 gccacaaaaa cggcccccaa agtccctggg acgtctccca gggttgcggc cgggtgttca
     3541 gaactcgtca gttccaccac gggtccgcca gatacagagc tagttagcta actagtacag
     3601 acgcaggcgc aaacattaga tgccggcaca aacacgcaca aagacagaga cagacacgaa
     3661 acgaccgctg gccagcttac ctcccggcgg gggggtcggt ggtccctggg caggggtctc
     3721 caaatctcgg tggaacctcc aaatgaaaga cccccgaggt gggcagtcaa tcaatctgag
     3781 gagaccctcc caaggatcag cgagtccacg attcggatgc aaacagcaag aggctttatt
     3841 gggaatacgg gtacccgggc gacgcagtct atcggaagac tggcgcgccg agtgaggggt
     3901 tgtgggctct tttattgagc tcggagagcg gaagcgcgcg aacagaagcg agaagcgaac
     3961 tgattggtta gttcaaataa ggtacagggt cattttcagg tccttggggc accctggaaa
     4021 catctgatga ttcactagaa actgctgagg gctggaccgc atctggggac catctgttct
     4081 tggccccgag ccggggcagg aactgcttac cacagatatc ctgtttggcc catcactcag
     4141 ctgtctcatc tgttcttggc cctgagccgg ggcaggaacc gcttaccaca gatatcctgt
     4201 ttggtattca gctgtttctt tgttcctgac cttgatctga acttttctat tctcagttat
     4261 gtatttttcc atgccttgca aagtggcgtt acttaagcta gcttgccacc tacgggtggg
     4321 gtctttcaaa agcaaagaaa cggaagggcc gcggcggggg ggcccggtac ccaattcgcc
     4381 ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4441 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4501 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4561 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4621 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4681 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4741 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4801 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4861 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4921 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4981 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     5041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5161 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5221 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5281 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5341 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5401 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5461 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5521 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5581 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5641 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5701 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5761 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5821 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5881 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5941 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6001 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6841 ttatcccc
//
