LOCUS       sequence-154081-        6336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expressing a single sgRNA with Scaffold MS2 tail, under 
            U6:3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6336)
  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 6336)
  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-Scaffold
FEATURES             Location/Qualifiers
     source          1..6336
                     /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        538..556
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(912..932)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(912..930)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(951..967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(951..967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(964..986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    975..991
                     /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(999..1005,1006..1023,1024..1029))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1044..1065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1182..1199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1353..1941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1433..1452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2112..2903,2904..2972))
                     /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     2735..2754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2973..3077)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3103..3558)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3240..3261)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3452..3471
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3685..3707
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3699..3716
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3700..3716
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3723..3742
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3723..3741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3933..4050,4127..4220,4312..4918,4973..5106,
                     5167..5327,5401..5426))
                     /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    5741..5810
                     /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 gacctgttta agagctatgc tggaaacagc atagcaagtt
      481 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cgggagcaca
      541 tgaggatcac ccatgtgcga ctcccacagt cactggggag tcttcccttt ttttgttttt
      601 tttttttgcc tacctggagc ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt
      661 gctttcgcca gtatttatta tttttcatca atatgtattc aatttggtat gtatttagta
      721 attgtaatat atagacaatg gttttccgtt gacgtacata catctgacgt gtgtttattt
      781 agacataata gttatgtttt cacatctttt taatgttcgc ttaatgcgta tgcattctag
      841 acaattgtgc tcggcaacag tatatttgtg gtgtgccaac caacaacctg caggagctcc
      901 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
      961 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     1021 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     1081 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1141 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     1201 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1261 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1321 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     1381 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     1441 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     1501 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     1561 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1621 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1681 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1741 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     1801 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     1861 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1921 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1981 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2041 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2101 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2161 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2221 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2281 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2341 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2401 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2461 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2521 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2581 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2641 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2701 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2761 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2821 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2881 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2941 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3001 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3061 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     3121 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3181 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3241 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3301 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     3361 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3421 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3481 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3541 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     3601 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     3661 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     3721 tgtaatacga ctcactatag ggcgaattgg gtacaagctt ggatttattt tgttatgtta
     3781 tatgtattat atgtcagaca taaagaaaag gaacacatca aatgtgataa caaagactaa
     3841 acaagtaatt ttattacacc aaaacgacaa aacagtaggc agaacaaaca acgcatagcc
     3901 aaacattgac gaattggata ccctgccgat tgtcagacac ttttgttgat cagtttcttg
     3961 cgaatggtct cgtccagcgg tggaatcgcc tcgcggggaa tcagaaaagt ggacagattg
     4021 aacagatcca gaaacacctt gtaccgatca ctgaaaccaa aaaaaaacaa agggagaaca
     4081 gtttgagttc attgatcccc gatataatca catctgcgat gatcacctga gagtggagcg
     4141 cagatattga tatccagacg agccaccagt gcccaactgt tgggatccaa tcatgcgttg
     4201 caccatgatc acgtgattgt ctgcggcggg aatagaaagt atttggttag gaaaaccagt
     4261 cttaaacata agatatattt ataaaagagt atcaaagaat gcaatactta catctccact
     4321 tggttattaa cgagtcgatg tccatgagca gggtgagcaa ctggtgtggt tggctgaacc
     4381 tgggttcatc cctatagaag gtgatcatga tggctccctg aagggcacga tggctaaacc
     4441 ggcgatcccc acgacgcacc agtgcatcgt gcactgccgg atcaaagatg gagcgataca
     4501 cctcgcgtcg cttctcaatg tccatgaggc ggtagttttt cgccttctcc acgggctcct
     4561 ccatggcgct ctgtacctgc gcctccagga atcgatcgac gctctcctga aacttggccc
     4621 agaagttgaa gccactctcc tccagtccgg gcgtcctctc cagccatcgc tgcactagct
     4681 ccagtagcga gggatctttc tccgagttgc gaatcgagtt ccgcgcctcc tcgtcgctaa
     4741 agacatccga gtacttctgg ttgtatctca cccgctgctc tgtcagaact cccagcttgt
     4801 tctcgatcaa acggaactgc agcgactgaa aaccagatgc gggtgccagg tacttgcgga
     4861 agtccatgaa gtctagcggg gtcatggtct ccagaatggg cacttggtcc accaggagct
     4921 gtacaaagga agttataaac ggattttggt aagagattca gaaagcactc acttttagaa
     4981 tcagaaccac tcggttcagt cgcttgacaa tctccagcgt cttggtttca tcgatgacct
     5041 ctgcatccaa catgtctcgt atggagtcga actcaaagat gatctgcttg aaccaaagct
     5101 cgtaggctgt ggcgaaggta cttaaatgcc attgagtgtt gtcatcaaag ttgtaaacct
     5161 actcaccctg gtgcgtgatg atgaacagat gctcatcgtg cacgggtcgc ttgtcctcct
     5221 cggacagcat acactgggca tccagcagtt tgtccagcat cagatactct ccatagattt
     5281 tgcccacttc cgtggttaat ggcaccgccg aatcatcgtg atcgtttctg tatgggtttg
     5341 aattgaatcg cagaactgaa gatcgattgg cattcctgga cagcacgtgc tggtgctcac
     5401 ccgtttcctg catagggaca gctcatggtg cacagctcag atcagatcgt gactcctcga
     5461 gcggcggatg ctggcgaact gatctccgcc agcggaccgg agatgagacc ccagcgaacc
     5521 gataacagag cgagagagct ccagttccga ctgattgcac agtcggtgat ctgggcgatg
     5581 ggcactgcca gataggctgg gaattatcaa tcacttgagg tgaaagtgcg gcgcacacaa
     5641 atccaagctt gatatcatcg atctcgaggc tgcatccaac gcgttgggag ctctccggat
     5701 caattcggct tcaggtaccg tcgacgatgt aggtcacggt ctcgaagccg cggtgcgggt
     5761 gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc tggtccatca
     5821 tgatgaacgg gtcgaggtgg cggtagttga tcccggcgaa cgcgcggcgc accgggaagc
     5881 cctcgccctc gaaaccgctg ggcgcggtgg tcacggtgag cacgggacgt gcgacggcgt
     5941 cggctgggtg cggatacgcg gggcagcgtc agcgggttct cgacggtcac ggcgggcatg
     6001 tcgacaagcc gaattgatcc actagaaggc ctaattcggt acactagttg gccacgtaat
     6061 aagtgtgcgt tgaatttatt cgcaaaaaca ttgcatattt tcggcaaagt aaaattttgt
     6121 tgcatacctt atcaaaaaat aagtgctgca tactttttag agaaaccaaa taatttttta
     6181 ttgcataccc gtttttaata aaatacattg cataccctct tttaataaaa aatattgcat
     6241 actttgacga aacaaatttt cgttgcatac ccaataaaag attattatat tgcatacctt
     6301 ttcttgccat accatttagc cgatcaattg agatct
//
