LOCUS       sequence-154078-        6317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expressing a single sgRNA with SAM MS2 tail, under U6:3 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6317)
  AUTHORS   Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan
            D, Haque SJ, Cecchi RJ, Kowal EJ, Buchthal J, Housden BE, Perrimon 
            N, Collins JJ, Church G
  TITLE     Comparison of Cas9 activators in multiple species.
  JOURNAL   Nat Methods. 2016 May 23. doi: 10.1038/nmeth.3871.
  PUBMED    27214048
REFERENCE   2  (bases 1 to 6317)
  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"; journalName: "Nat 
            Methods. 2016 May 23. doi: 10.1038/nmeth.3871."
COMMENT     Sequence Label: pCFD3-MS2tail-SAM
FEATURES             Location/Qualifiers
     source          1..6317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..427
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        463..481
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        533..551
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(892..912)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(892..910)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(931..947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(931..947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(944..966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    955..971
                     /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(979..985,986..1003,1004..1009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1024..1045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1162..1179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1333..1921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1413..1432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2092..2883,2884..2952))
                     /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     2715..2734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2953..3057)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3083..3538)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3220..3241)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3432..3451
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3665..3687
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3679..3696
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3680..3696
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3703..3722
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3703..3721
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3913..4030,4107..4200,4292..4898,4953..5086,
                     5147..5307,5381..5406))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the brown 
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    5721..5790
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
       61 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
      121 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
      181 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
      241 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
      301 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
      361 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
      421 aacgtcgggg tcttcgagaa gacctgtttt agagctaggc caacatgagg atcacccatg
      481 tctgcagggc ctagcaagtt aaaataaggc tagtccgtta tcaacttggc caacatgagg
      541 atcacccatg tctgcagggc caagtggcac cgagtcggtg cttttttgcc tacctggagc
      601 ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt gctttcgcca gtatttatta
      661 tttttcatca atatgtattc aatttggtat gtatttagta attgtaatat atagacaatg
      721 gttttccgtt gacgtacata catctgacgt gtgtttattt agacataata gttatgtttt
      781 cacatctttt taatgttcgc ttaatgcgta tgcattctag acaattgtgc tcggcaacag
      841 tatatttgtg gtgtgccaac caacaacctg caggagctcc agcttttgtt ccctttagtg
      901 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      961 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     1021 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1141 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1501 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1741 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     1801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1981 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     2041 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2101 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2161 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2221 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2281 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2341 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2401 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2461 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2521 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2581 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2641 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2701 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2761 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2821 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2881 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2941 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     3001 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3061 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     3121 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3181 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3241 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3301 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3361 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3421 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3481 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     3541 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3601 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3661 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     3721 ggcgaattgg gtacaagctt ggatttattt tgttatgtta tatgtattat atgtcagaca
     3781 taaagaaaag gaacacatca aatgtgataa caaagactaa acaagtaatt ttattacacc
     3841 aaaacgacaa aacagtaggc agaacaaaca acgcatagcc aaacattgac gaattggata
     3901 ccctgccgat tgtcagacac ttttgttgat cagtttcttg cgaatggtct cgtccagcgg
     3961 tggaatcgcc tcgcggggaa tcagaaaagt ggacagattg aacagatcca gaaacacctt
     4021 gtaccgatca ctgaaaccaa aaaaaaacaa agggagaaca gtttgagttc attgatcccc
     4081 gatataatca catctgcgat gatcacctga gagtggagcg cagatattga tatccagacg
     4141 agccaccagt gcccaactgt tgggatccaa tcatgcgttg caccatgatc acgtgattgt
     4201 ctgcggcggg aatagaaagt atttggttag gaaaaccagt cttaaacata agatatattt
     4261 ataaaagagt atcaaagaat gcaatactta catctccact tggttattaa cgagtcgatg
     4321 tccatgagca gggtgagcaa ctggtgtggt tggctgaacc tgggttcatc cctatagaag
     4381 gtgatcatga tggctccctg aagggcacga tggctaaacc ggcgatcccc acgacgcacc
     4441 agtgcatcgt gcactgccgg atcaaagatg gagcgataca cctcgcgtcg cttctcaatg
     4501 tccatgaggc ggtagttttt cgccttctcc acgggctcct ccatggcgct ctgtacctgc
     4561 gcctccagga atcgatcgac gctctcctga aacttggccc agaagttgaa gccactctcc
     4621 tccagtccgg gcgtcctctc cagccatcgc tgcactagct ccagtagcga gggatctttc
     4681 tccgagttgc gaatcgagtt ccgcgcctcc tcgtcgctaa agacatccga gtacttctgg
     4741 ttgtatctca cccgctgctc tgtcagaact cccagcttgt tctcgatcaa acggaactgc
     4801 agcgactgaa aaccagatgc gggtgccagg tacttgcgga agtccatgaa gtctagcggg
     4861 gtcatggtct ccagaatggg cacttggtcc accaggagct gtacaaagga agttataaac
     4921 ggattttggt aagagattca gaaagcactc acttttagaa tcagaaccac tcggttcagt
     4981 cgcttgacaa tctccagcgt cttggtttca tcgatgacct ctgcatccaa catgtctcgt
     5041 atggagtcga actcaaagat gatctgcttg aaccaaagct cgtaggctgt ggcgaaggta
     5101 cttaaatgcc attgagtgtt gtcatcaaag ttgtaaacct actcaccctg gtgcgtgatg
     5161 atgaacagat gctcatcgtg cacgggtcgc ttgtcctcct cggacagcat acactgggca
     5221 tccagcagtt tgtccagcat cagatactct ccatagattt tgcccacttc cgtggttaat
     5281 ggcaccgccg aatcatcgtg atcgtttctg tatgggtttg aattgaatcg cagaactgaa
     5341 gatcgattgg cattcctgga cagcacgtgc tggtgctcac ccgtttcctg catagggaca
     5401 gctcatggtg cacagctcag atcagatcgt gactcctcga gcggcggatg ctggcgaact
     5461 gatctccgcc agcggaccgg agatgagacc ccagcgaacc gataacagag cgagagagct
     5521 ccagttccga ctgattgcac agtcggtgat ctgggcgatg ggcactgcca gataggctgg
     5581 gaattatcaa tcacttgagg tgaaagtgcg gcgcacacaa atccaagctt gatatcatcg
     5641 atctcgaggc tgcatccaac gcgttgggag ctctccggat caattcggct tcaggtaccg
     5701 tcgacgatgt aggtcacggt ctcgaagccg cggtgcgggt gccagggcgt gcccttgggc
     5761 tccccgggcg cgtactccac ctcacccatc tggtccatca tgatgaacgg gtcgaggtgg
     5821 cggtagttga tcccggcgaa cgcgcggcgc accgggaagc cctcgccctc gaaaccgctg
     5881 ggcgcggtgg tcacggtgag cacgggacgt gcgacggcgt cggctgggtg cggatacgcg
     5941 gggcagcgtc agcgggttct cgacggtcac ggcgggcatg tcgacaagcc gaattgatcc
     6001 actagaaggc ctaattcggt acactagttg gccacgtaat aagtgtgcgt tgaatttatt
     6061 cgcaaaaaca ttgcatattt tcggcaaagt aaaattttgt tgcatacctt atcaaaaaat
     6121 aagtgctgca tactttttag agaaaccaaa taatttttta ttgcataccc gtttttaata
     6181 aaatacattg cataccctct tttaataaaa aatattgcat actttgacga aacaaatttt
     6241 cgttgcatac ccaataaaag attattatat tgcatacctt ttcttgccca taccatttag
     6301 ccgatcaatt gagatct
//
