LOCUS       Exported                6075 bp ds-DNA     circular SYN 18-JUN-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6075)
  AUTHORS   Ryan Lister
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Oct 31, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6075
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     polyA_signal    6..230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6..27
                     /label=OC-310_pLenti_1_dCas9_5.F
     primer_bind     complement(204..230)
                     /label=OC-311_pLenti_1_dCas9_5.R
     primer_bind     complement(210..253)
                     /label=TOL827_pGHpolyA_R
     primer_bind     211..254
                     /label=TOL825_pEF1a-GFP_R
     CDS             complement(240..956)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     240..260
                     /label=TOL195_GFP_PUF_polyA_R
     primer_bind     complement(666..684)
                     /label=TOL102_GFP_backbone_F
     primer_bind     682..696
                     /label=TOL103_GFP_backbone_R
     primer_bind     complement(939..956)
                     /label=TOL194_T2A_GFP_F
     primer_bind     complement(949..988)
                     /label=TOL837_GFP_bGHpolyA_F
     primer_bind     949..987
                     /label=TOL840_pSV40_R
     promoter        complement(981..1191)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      987..1122
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1170..1224)
                     /label=TOL839_pSV40_F
     primer_bind     1183..1225
                     /label=TOL847_pEF1a_F
     primer_bind     complement(1212..1229)
                     /label=TOL847_pEF1a_F
     primer_bind     1213..1229
                     /label=TOL839_pSV40_F
     primer_bind     1291..1312
                     /label=TOL318_GCN4_pEF1a_F
     primer_bind     complement(1291..1312)
                     /label=TOL319_GCN4_pEF1a_R
     primer_bind     1331..1356
                     /label=OL-D206
     promoter        1406..2663
                     /label=EF1a
     primer_bind     complement(1478..1501)
                     /label=OL-D243
     primer_bind     1846..1867
                     /label=OL -D154
     primer_bind     2458..2479
                     /label=OL- D155
     primer_bind     2524..2545
                     /label=TOL417_GCN4_seq_F
     primer_bind     complement(2655..2674)
                     /label=TOL848_pEF1a_R
     misc_feature    2670..2675
                     
     primer_bind     complement(2673..2702)
                     /label=TOL401_3xTy1_GCN4_ R
     CDS             2676..2678
                     /codon_start=1
                     /label=ATG
                     /translation="M"
     CDS             2682..2702
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2703..2744
                     /codon_start=1
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2745..2786
                     /codon_start=1
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2787..2828
                     /codon_start=1
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     2827..2854
                     /label=TOL784_Traptavidin_F
     CDS             2832..3215
                     /codon_start=1
                     /label=Traptavidin?
                     /translation="MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAEGDYVL
                     TGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTT
                     EANAWKSTLVGHDTFTKVKPSAAS"
     primer_bind     complement(3191..3235)
                     /label=TOL787_Traptavidin_R2
     primer_bind     3196..3233
                     /label=TOL786_linker_p65HSF1_F3
     primer_bind     3215..3253
                     /label=TOL35
     CDS             3216..3269
                     /codon_start=1
                     /label=Linker
                     /translation="SAGGGGSGGGGSGGGGSG"
     primer_bind     3216..3235
                     /label=TOL774_linker_p65HSF1_F
     primer_bind     3238..3256
                     /label=TOL418_GB1linker_seq_F
     primer_bind     complement(3253..3269)
                     /label=TOL316_GCN4_p65HSF1_R2
     primer_bind     complement(3257..3278)
                     /label=TOL-29
     primer_bind     complement(3260..3278)
                     /label=TOL405
     misc_feature    3279..3566
                     /label=KRAB codon change
     primer_bind     3361..3380
                     /label=TOL457_KRAB_qPCR1_F
     primer_bind     complement(3440..3459)
                     /label=TOL458_KRAB_qPCR1_R
     misc_feature    complement(3570..3593)
                     /label=UPS3
     polyA_signal    3594..3832
                     /label=SV40 polyA
     primer_bind     3594..3633
                     /label=TOL400_UPS_polyA_F
     primer_bind     complement(3654..3678)
                     /label=TOL419_SV40_seq_R
     rep_origin      4311..4930
                     /label=pBR322 Orgin
     CDS             complement(5085..5945)
                     /codon_start=1
                     /label=Ampicillin
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5987..6015)
                     /label=AMP promoter
     primer_bind     join(6058..6075,1..25)
                     /label=TOL838_GFP_bGHpolyA_R
     primer_bind     complement(join(6060..6075,1..25))
                     /label=TOL846_p65HSF1_R
ORIGIN
        1 gacgtccata gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc
       61 cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga
      121 tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga
      181 aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcaactgccc
      241 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
      301 atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
      361 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
      421 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc
      481 ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc
      541 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
      601 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
      661 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
      721 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg
      781 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
      841 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
      901 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg
      961 gccgtacggg tggcgcgcgt agcctaggcc tccaaaaaag cctcctcact acttctggaa
     1021 tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg
     1081 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
     1141 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg cctgaattca
     1201 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     1261 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     1321 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     1381 ctgcttaggg ttaggcgttt tgcgtgcaaa gatggataaa gttttaaaca gagaggaatc
     1441 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     1501 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     1561 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     1621 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     1681 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     1741 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc
     1801 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
     1861 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
     1921 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
     1981 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
     2041 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
     2101 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
     2161 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
     2221 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     2281 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     2341 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     2401 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt
     2461 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     2521 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     2581 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     2641 ttcttccatt tcaggtgtcg tgacgtacgg ccaccatggg tcctaagaaa aagaggaagg
     2701 tgggtaagcc tatccctaac cctctcctcg gtctcgattc tacgggcaag cccatcccca
     2761 accccctgct gggcctggac agcaccggta aacctattcc taatccactc ctcggactcg
     2821 attccacaca tatggctgaa gctggtatca ccggcacctg gtacaaccag ctgggatcca
     2881 ccttcatcgt taccgctggt gctgacggtg ctctgaccgg tacctacgaa tccgctgttg
     2941 gtaacgctga aggcgattac gttctgaccg gtcgttacga ctccgctccg gctaccgacg
     3001 gttccggaac cgctctgggt tggaccgttg cttggaaaaa caactaccgt aacgctcact
     3061 ccgctaccac ctggtctggc cagtacgttg gtggtgctga agctcgtatc aacacccagt
     3121 ggttgttgac ctccggcacc accgaagcta acgcgtggaa atccaccctg gttggtcacg
     3181 acaccttcac caaagttaaa ccgtccgctg cttccagcgc tggagggggt ggctccggcg
     3241 gaggaggctc tggtggcgga gggagtggag cggccgctga tgccaagtcc ctgaccgcct
     3301 ggagccgcac cttggtaact tttaaggatg ttttcgttga ttttactaga gaggagtgga
     3361 aattgcttga caccgctcag cagattctgt accggaacgt tatgctggaa aattacaaga
     3421 atttggtgag tcttggctac caacttacta agcccgacgt tattttgaga ctcgagaagg
     3481 gggaggaacc gtggctggtg gaacgggaga ttcatcagga gacacatcct gattctgaaa
     3541 cagcttttga aataaagtct tctgtttgac gttgagtctc tgtttgtggt gactgatcat
     3601 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3661 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3721 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3781 gcattctagt tgtggtttgt ccaaactcat caatgtatct taatgcgtcg agtgcattct
     3841 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     3901 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3961 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4021 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4081 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4141 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4201 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4261 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4321 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4381 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4441 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4501 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4561 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4621 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4681 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4741 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4801 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4861 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4921 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4981 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5041 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5101 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5161 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5221 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5281 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5341 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5401 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5461 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5521 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5581 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5641 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5701 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5761 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5821 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5881 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5941 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6001 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6061 cgaaaagtgc cacct
//
