LOCUS       sequence-272380-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Goldengate/Greengate module for genome editing, using the SaCAS9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  TITLE     Theres lab:  Alternative Greengate/Goldengate modules
REFERENCE   2  (bases 1 to 7348)
  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"
COMMENT     Sequence Label: pGGA_RT_Sa_Cas9
FEATURES             Location/Qualifiers
     source          1..7348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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        2212..2230
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2212..2229
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3236..6394
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN
                     LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLL
                     NRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQ
                     IIKKG"
     CDS             6407..6427
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6934..6953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6935..6953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6963..6985)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7179..7198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7298..7320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtggctct cgaggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacgagc tatttaggtg
     2221 acactataga agtgaagctt ggtctcaacc taaaaattac ggatatgaat ataggcatat
     2281 ccgtatccga attatccgtt tgacagctag caacgattgt acaattgctt ctttaaaaaa
     2341 ggaagaaaga aagaaagaaa agaatcaaca tcagcgttaa caaacggccc cgttacggcc
     2401 caaacggtca tatagagtaa cggcgttaag cgttgaaaga ctcctatcga aatacgtaac
     2461 cgcaaacgtg tcatagtcag atcccctctt ccttcaccgc ctcaaacaca aaaataatct
     2521 tctacagcct atatatacaa cccccccttc tatctctcct ttctcacaat tcatcatctt
     2581 tctttctcta cccccaattt taagaaatcc tctcttctcc tcttcatttt caaggtaaat
     2641 ctctctctct ctctctctct gttattcctt gttttaatta ggtatgtatt attgctagtt
     2701 tgttaatctg cttatcttat gtatgcctta tgtgaatatc tttatcttgt tcatctcatc
     2761 cgtttagaag ctataaattt gttgatttga ctgtgtatct acacgtggtt atgtttatat
     2821 ctaatcagat atgaatttct tcatattgtt gcgtttgtgt gtaccaatcc gaaatcgttg
     2881 atttttttca tttaatcgtg tagctaattg tacgtataca tatggatcta cgtatcaatt
     2941 gttcatctgt ttgtgtttgt atgtatacag atctgaaaac atcacttctc tcatctgatt
     3001 gtgttgttac atacatagat atagatctgt tatatcattt tttttattaa ttgtgtatat
     3061 atatatgtgc atagatctgg attacatgat tgtgattatt tacatgattt tgttatttac
     3121 gtatgtatat atgtagatct ggactttttg gagttgttga cttgattgta tttgtgtgtg
     3181 tatatgtgtg ttctgatctt gatatgttat gtatgtgcag cgaattcggc gcgccatgaa
     3241 gaggaactac atcctcggac tcgatatcgg aatcacctct gtgggatacg gaatcatcga
     3301 ttacgagact agggatgtga tcgatgctgg tgtgagactc ttcaaagagg ctaacgttga
     3361 gaacaacgag ggaagaaggt ctaagagggg agctagaagg ctcaagagaa gaagaaggca
     3421 cagaatccag agggtgaaga agctcctctt cgattacaac ctcctcaccg atcactctga
     3481 gctttctgga atcaaccctt acgaggcaag agtgaaggga ctctctcaga agttgtctga
     3541 ggaagagttc tctgctgctt tgctccacct tgctaagaga aggggtgtgc ataacgtgaa
     3601 cgaggtggaa gaggataccg gaaacgagct ttctaccaaa gagcagatct ctaggaactc
     3661 taaggctctc gaagagaagt acgtggcaga gcttcagctt gagagactca agaaagatgg
     3721 tgaggtgagg ggatctatca acaggttcaa gacctctgat tacgtgaaag aagctaagca
     3781 gctcctcaag gtgcagaagg cttaccatca gctcgatcag tctttcatcg atacctacat
     3841 cgatcttctc gagactagaa ggacctacta cgagggacct ggtgaaggat ctccattcgg
     3901 atggaaggat atcaaagaat ggtacgagat gctcatggga cactgcactt acttccctga
     3961 agaactcaga tctgtgaagt acgcttacaa cgctgatctc tacaacgctc ttaacgatct
     4021 caacaacctc gtgatcacca gggatgagaa cgagaagctt gagtactacg agaagttcca
     4081 gatcatcgag aacgtgttca agcaaaagaa gaagcctacc ctcaagcaga tcgctaaaga
     4141 gatccttgtt aacgaggaag atatcaaggg atacagggtg acctctaccg gaaagcctga
     4201 gttcactaac ctcaaggttt accacgatat taaggatatt accgctagaa aagagattat
     4261 tgagaacgct gagcttctcg atcaaatcgc taagatcctt accatctacc agtcatctga
     4321 ggatatccaa gaggaattga ccaacctcaa ctcagagctt acccaagaag agatcgagca
     4381 gatcagtaac ttgaagggtt acaccggaac ccacaacctc tcactcaagg ctatcaacct
     4441 catcctcgat gagctttggc acaccaacga taaccagatc gcaatcttca acagactcaa
     4501 gctcgtgcct aagaaagtgg atctctctca gcagaaagag attcctacca ccctcgtgga
     4561 tgatttcatc ctctcacctg ttgtgaagag atctttcatc cagtctatca aggtgatcaa
     4621 cgctattatc aagaaatacg gactccctaa cgatatcatc atcgagttgg ctagggaaaa
     4681 gaactcaaag gatgctcaaa agatgatcaa cgagatgcag aagagaaaca ggcagaccaa
     4741 cgagaggatc gaagagatca tcaggaccac cggaaaagag aacgctaagt acctcattga
     4801 gaagatcaag ctccacgata tgcaagaggg aaagtgcctc tactctctcg aggctatccc
     4861 tcttgaggat ctccttaaca accctttcaa ctacgaagtg gatcacatca tccctagatc
     4921 tgtgtctttc gataactctt tcaacaacaa ggtgctcgtg aagcaagagg aaaactctaa
     4981 gaagggtaac aggacccctt tccagtacct ctcttcatct gattctaaga tctcatacga
     5041 gactttcaag aagcacatcc tcaacctcgc taagggaaag ggtaggatct ctaagacaaa
     5101 gaaagagtac cttctcgagg aaagggatat caacagattc tctgttcaga aggatttcat
     5161 caacagaaac cttgtggata ccaggtacgc taccaggggt cttatgaacc ttctcaggtc
     5221 ttacttcagg gtgaacaacc ttgatgtgaa ggtgaagtca atcaacggtg gattcacctc
     5281 attccttaga agaaaatgga agtttaagaa agagaggaac aagggttata agcaccacgc
     5341 tgaggatgct ctcattatcg ctaacgcaga tttcattttc aaagagtgga agaagttgga
     5401 taaggctaaa aaggtgatgg aaaaccagat gttcgaggaa aagcaggctg agtctatgcc
     5461 tgagatcgag actgagcaag agtacaaaga aatcttcatc acccctcacc agatcaagca
     5521 catcaaagat ttcaaggatt acaagtactc tcacagagtg gataagaagc caaacagaga
     5581 gcttatcaac gataccctct actcaaccag aaaggatgat aagggaaaca ccctcatcgt
     5641 gaacaatctc aacggactct acgataagga taacgataag ctcaagaagc tcattaacaa
     5701 gtctccagag aagttgctca tgtaccacca cgatcctcag acctaccaaa agctcaagct
     5761 catcatggaa cagtacggtg atgagaagaa ccctctctac aagtattacg aggaaaccgg
     5821 taactacctc accaagtact caaaaaagga taatggacct gtgatcaaga agattaagta
     5881 ctacggaaac aagctcaacg cacacctcga tatcaccgat gattacccta acagtagaaa
     5941 caaggtggtg aagctttcac tcaagcctta caggttcgat gtgtacctcg ataacggtgt
     6001 gtacaagttc gtgaccgtga agaacctcga tgttatcaaa aaagaaaact actatgaggt
     6061 taactctaag tgctacgagg aagctaagaa gttgaagaag atctctaacc aggcagagtt
     6121 cattgcttca ttctacaaca acgatcttat caagattaac ggtgagcttt acagggttat
     6181 cggagtgaac aacgatttgc tcaacaggat cgaggtgaac atgattgata tcacctacag
     6241 ggaatacctc gagaatatga acgataagag gcctcctaga atcatcaaga ctatcgcttc
     6301 taagacccag agtatcaaga agtactctac cgatatcctc ggaaacctct acgaggttaa
     6361 gtcaaagaag caccctcaga tcattaagaa gggatcaagg gctgatccta agaagaagag
     6421 gaaggtttga ggcgcgccga gctccaggcc tcccagcttt cgtccgtatc atcggtttcg
     6481 acaacgttcg tcaagttcaa tgcatcagtt tcattgccca cacaccagaa tcctactaag
     6541 tttgagtatt atggcattgg aaaagctgtt ttcttctatc atttgttctg cttgtaattt
     6601 actgtgttct ttcagttttt gttttcggac atcaaaatgc aaatggatgg ataagagtta
     6661 ataaatgata tggtcctttt gttcattctc aaattattat tatctgttgt ttttacttta
     6721 atgggttgaa tttaagtaag aaaggaacta acagtgtgat attaaggtgc aatgttagac
     6781 atataaaaca gtctttcacc tctctttggt tatgtcttga attggtttgt ttcttcactt
     6841 atctgtgtaa tcaagtttac tatgagtcta tgatcaagta attatgcaat caagttaagt
     6901 acagtatagg cttaacatga gaccgaattc tcgccctata gtgagtcgta ttacctggcc
     6961 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     7021 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     7081 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     7141 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     7201 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     7261 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     7321 ttttcaccgt catcaccgaa acgcgcga
//
