LOCUS       sequence-417251-        7490 bp    DNA     circular SYN 15-APR-2025
DEFINITION  The SSA-based surrogate reporter for validating the activity of 
            different gene editing tools as well as for helping to screen 
            gene-editing positive cells. Key Construct: 
            CMV-DsRed-CMV-eGFP*(SSA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7490)
  AUTHORS   Xu K, Ren C, Liu Z, Zhang T, Zhang T, Li D, Wang L, Yan Q, Guo L, 
            Shen J, Zhang Z
  TITLE     Efficient genome engineering in eukaryotes using Cas9 from 
            Streptococcus thermophilus.
  JOURNAL   Cell Mol Life Sci. 2015 Jan;72(2):383-99. doi: 
            10.1007/s00018-014-1679-z. Epub 2014 Jul 20.
  PUBMED    25038777
REFERENCE   2  (bases 1 to 7490)
  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"; doi: 
            "10.1007/s00018-014-1679-z"; journalName: "Cell Mol Life Sci"; date:
            "2015-01"; volume: "72"; issue: "2"; pages: "383-99"
COMMENT     Sequence Label: pSSA-DsRed/eGFP (pXK-442)
FEATURES             Location/Qualifiers
     source          1..7490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   2335..2369
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2748..3135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3136..3339
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3373..3382
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3379..3381,3382..3384,3385..4059)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(3562..3582)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3780..3799
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3974..3997
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4063..4082)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4128..4183
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4182..4201)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        4592..4895
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4896..5099
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      5138..5147
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5189..5210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5698..5717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6045..6066
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6247..6368
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6284..6303)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6338..6357
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6718..6780
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6846..6865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6847..6865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6872..6889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6872..6888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6881..6903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7030..7485
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7117..7136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7327..7348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctggt acctcgcgcg acttggtttg
     2221 ccattcttta gcgcgcgtcg cgtcacacag cttggccaca atgtggtttt tgtcaaacga
     2281 agattctatg acgtgtttaa agtttaggtc gagtaaagcg caaatctttt ttaaccctag
     2341 aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc tctttctaaa
     2401 tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga gctcccgtga
     2461 ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc gcgtgagtca
     2521 aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg ataattatat
     2581 tgttatttca tgttctactt acgtgataac ttattatata tatattttct tgttatagat
     2641 atcgtgacta atatataata aaatgggtag ttctttagac gatgagcata tcctctctgc
     2701 tcttctgcaa agcgatgacg agcttgttgg ctagcggtgg cggcctcgac attgattatt
     2761 gactagagca ttagttatta atagtaatca attacggggt cattagttca tagcccatat
     2821 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     2881 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     2941 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     3001 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     3061 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     3121 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     3181 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     3241 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     3301 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
     3361 cgctagccgg tcgccaccat ggtgcgctcc tccaagaacg tcatcaagga gttcatgcgc
     3421 ttcaaggtgc gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc
     3481 gagggccgcc cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc
     3541 ctgcccttcg cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg
     3601 aagcaccccg ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg
     3661 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     3721 caggacggct gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc
     3781 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc
     3841 gacggcgtgc tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac
     3901 ctggtggagt tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac
     3961 tacgtggact ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag
     4021 tacgagcgca ccgagggccg ccaccacctg ttcctgtaag gatctttttc cctctgccaa
     4081 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     4141 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     4201 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     4261 ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca
     4321 gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt
     4381 tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt
     4441 actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt
     4501 atggagatcc ctcgacctgc agcccaagct ttagttatta atagtaatca attacggggt
     4561 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     4621 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     4681 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     4741 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     4801 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     4861 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     4921 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     4981 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     5041 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     5101 gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatggtga gcaagggcga
     5161 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5221 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     5281 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     5341 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     5401 gtccgccatg cccgaaggct acgtccagga gcgcagcggc cgcgaattcg gggatcctaa
     5461 gtgactaaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     5521 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     5581 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     5641 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     5701 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     5761 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     5821 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     5881 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     5941 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     6001 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     6061 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga
     6121 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     6181 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     6241 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     6301 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     6361 gtatcttact cgagttaatt aacgagagca taatattgat atgtgccaaa gttgtttctg
     6421 actgactaat aagtataatt tgtttctatt atgtataggt taagctaatt acttatttta
     6481 taatacaaca tgactgtttt taaagtacaa aataagttta tttttgtaaa agagagaatg
     6541 tttaaaagtt ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata
     6601 aataaacata aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata
     6661 aaacttaata tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat
     6721 gcgtcaattt tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg
     6781 ttaaataata gtttctaatt tttttattat tcagcctgct gtcgtgaata ccgagctcca
     6841 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     6901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     6961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     7021 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     7081 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     7141 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     7201 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     7261 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7321 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     7381 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     7441 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
//
