LOCUS       Exported                6856 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    pPRISM-TagRFP-cmlc2-mRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6856)
  AUTHORS   Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Weiss TJ, 
            Torrie ME, Vollbrecht MK, Lan M, McKeighan KC, Ming Z, Wehmeier A, 
            Mikelson CS, Haltom JA, Kwan KM, Chien CB, Balciunas D, Ekker SC, 
            Clark KJ, Webber BR, Moriarity B, Solin SL, Carlson DF, Dobbs DL, 
            McGrail M, Essner JJ.
  TITLE     GeneWeld: a method for efficient targeted integration directed by 
            short homology
  JOURNAL   bioRxiv 431627
REFERENCE   2  (bases 1 to 6856)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6856
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    532..553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        568..598
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             642..1007
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSSRGV"
     primer_bind     665..685
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        667..685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(761..778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(770..792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1000..1710
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3473..3493
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3497..4174
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3627..3645)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3895..3914
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4089..4112
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4184..4408
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4478..4559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4683..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5081..5099
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5375..5394)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5386..6195
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5453..5472)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6338..70
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6827..6846
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt cccggagcca cgaacttctc tctgttaaag
      961 caagcaggag acgtggaaga aaaccccggt cctctcgagg tgtctaaggg cgaggaactg
     1021 attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc
     1081 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     1141 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
     1201 ggcagcagaa ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
     1261 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     1321 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     1381 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccaacacc
     1441 gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc cctgaagctc
     1501 gtgggcgggg gccacctgat ctgcaacttc aagaccacat acagatccaa gaaacccgct
     1561 aagaacctca agatgcccgg cgtctactat gtggaccaca gactggaaag aatcaaggag
     1621 gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc
     1681 cctagcaaac tggggcacaa acttaattga aactagtaat taagtctcag ccaccgttaa
     1741 ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa gataaagcct
     1801 ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt gctacatggc
     1861 tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact ctccttctca
     1921 cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac accacacact
     1981 gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga catatttata
     2041 tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt tctgcattga
     2101 aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa tttcagcaaa
     2161 caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta ctactttgca
     2221 gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac tttgacaaac
     2281 acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata tattatatat
     2341 atatatatat catataataa tattcagtat aatatatata tatatatcat attataatat
     2401 tcagtataat ataaaacaca aacacatata tgtataatat aatataacat ttttatttat
     2461 tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta aacatgaagt
     2521 gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt aatttaccat
     2581 ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga ggacaactgt
     2641 agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct atgactctat
     2701 aaatatatat atattttttt tttataaaat gatttattta taactatata tccatttctc
     2761 agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg tccccgttgt
     2821 ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa cctgtctggt
     2881 gtacagctac cagagaagct tgagatccta gtcaccggcg gcattcatcc atccttttca
     2941 tccctcaaat ctctcattca cgtccccctc cccatctgca cactttatct cattttccac
     3001 cctgctggaa tctgagcact tgtgcagtta tcagggctcc tgtatttagg aggctctggg
     3061 tgtccatgta ggggacgaac agaaacactg cagaccttta tagaagaaca aatgataaga
     3121 gtcctcatac ataaagactc cattagaaac gtcagtgacc caggagccca gaccaacagc
     3181 aaagcagaca gtgaaggatc cacagcctga ctaactcgac aaaccccccc aaacctaagg
     3241 tgagttgatc tttaagcttt ttacattttc agctcgcata tatcaattcg cgcgcattgt
     3301 cacactagca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc
     3361 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc
     3421 ggttttctca ttcatttaca gttcagccgg aattcacctc gagaccatgg ctccaaagaa
     3481 gaagcgtaag gtaaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa
     3541 ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     3601 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     3661 cttcgcctgg gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca
     3721 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     3781 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     3841 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     3901 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg
     3961 cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc
     4021 cgaggtcaag accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac
     4081 cgacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     4141 gcgcgccgag ggccgccact ccaccggcgc ctaaagctgt gatctgtgcc ttctagttgc
     4201 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4261 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4321 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4381 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa agggcgaatt
     4441 aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     4501 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     4561 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     4621 aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac cacaactaga
     4681 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4741 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4801 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattca
     4861 gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac gttgtctagc
     4921 aaggaagtga agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc
     4981 aggtggtaat gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt
     5041 ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg
     5101 agaccaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat
     5161 aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa
     5221 aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt
     5281 acagtaatca agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga
     5341 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     5401 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5461 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     5521 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5581 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5641 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     5701 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5761 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5821 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5881 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5941 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     6001 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     6061 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     6121 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     6181 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg
     6241 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6301 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6361 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6421 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6481 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6541 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6601 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6661 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6721 agcgtgagct atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg
     6781 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6841 atctttatag tcctgt
//
