LOCUS       Exported                6991 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-gcry-TagBFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  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 6991)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6991
                     /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             3584..3604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3608..4309
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4319..4543
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4613..4694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4818..4899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5216..5234
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5510..5529)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5521..6330
                     /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(5588..5607)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6473..70
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6962..6981
                     /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 ttcctagttc tttaaactgt
     2941 tacaaagtgt ttggaccagc aactgaattg cactgtaaat taccacatca gggtctccgg
     3001 ggtcaacgct gctaagcaat ggactggaaa atttgtgaca ttgagttttt ggaataactg
     3061 ggaatgctgg ataactcttt gggtaaagct atggaacaaa agtaccagca tgaactaaga
     3121 aatgactgtc acagcacaac atcaaacaca atgcaagaca aaggttctct gcttttgcct
     3181 tacttgacag aaaaagtaag ctctaggttc atcgcatgca gacagcagca atacatgaca
     3241 tatataaagg ctggctcttt tctcccttcc attgaactga aacttccact cagtcagact
     3301 tgcgggggat ccacagcctg actaactcga caaacccccc caaacctaag gtgagttgat
     3361 ctttaagctt tttacatttt cagctcgcat atatcaattc gcgcgcattg tcacactagc
     3421 acccactagc ggtcagactg cagattgcag cacgaaacag gaagctgact ccacatggtc
     3481 acatgctcac tgaagtgttg acttccctga cagctgtgca ctttctaaac cggttttctc
     3541 attcatttac agttcagccg gaattcacct cgagaccatg gctccaaaga agaagcgtaa
     3601 ggtaaccatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3661 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3721 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3781 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     3841 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     3901 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     3961 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     4021 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     4081 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
     4141 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     4201 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     4261 ggccagatac tgcgacctcc ctagcaaact ggggcacaag ctcaattaaa gctgagatct
     4321 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4381 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4441 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4501 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4561 accaaagggc gaattaattc attgatgagt ttggacaaac cacaactaga atgcagtgaa
     4621 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     4681 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     4741 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattga tgagtttgga
     4801 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4861 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4921 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4981 aaatgtggtc attcagatca attaaccctc acccgggcta gcgatgcatg ggagctctga
     5041 cgtacgttgt ctagcaagga agtgaagagc tatccggata tatatgtatg ttatcataac
     5101 tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg taggcacgac
     5161 attgaatatg ctcttctccg ctgtggcccg aacgcctccc gctagcgatg ggatctaata
     5221 cgactcacta tagggagacc aagggaaaat agaatgaagt gatctccaaa aaataagtac
     5281 tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta
     5341 attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa
     5401 aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca cctctgggat
     5461 ccagctgcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     5521 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     5581 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     5641 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     5701 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     5761 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5821 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5881 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5941 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     6001 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     6061 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     6121 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     6181 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     6241 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     6301 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     6361 caaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6421 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6481 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6541 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6601 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6841 aactgagata cctacagcgt gagctatgag aaagcgccac ggttcccgaa gggagaaagg
     6901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6961 ggggaaacgc ctggtatctt tatagtcctg t
//
