LOCUS       Exported                6256 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    pPRISM-Stop-gcry-eGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  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 6256)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6256
                     /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..956
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSIMIGSND
                     "
     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             2836..2856
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2860..3579
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2860..3576
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2905..2926)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3166..3185)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3513..3534
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3584..3808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3878..3959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4083..4164
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4481..4499
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4775..4794)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4786..5595
                     /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(4853..4872)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5738..70
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6227..6246
                     /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 ctataatgat aggtagtaat gattagtaat
      961 gagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg
     1021 ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg
     1081 cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg
     1141 tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca
     1201 gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag
     1261 tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat
     1321 ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg
     1381 agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa
     1441 tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt attccaacac
     1501 gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc
     1561 tcacaggcaa tttagtataa tatattatat atatatatat atcatataat aatattcagt
     1621 ataatatata tatatatatc atattataat attcagtata atataaaaca caaacacata
     1681 tatgtataat ataatataac atttttattt attgagatgc ctctatggac cgtgttataa
     1741 gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa
     1801 gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac
     1861 caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac tttgctgaat
     1921 atagctggct tgacagttat ctatgactct ataaatatat atatattttt tttttataaa
     1981 atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc
     2041 gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca
     2101 tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc
     2161 tagtcaccgg cgttcctagt tctttaaact gttacaaagt gtttggacca gcaactgaat
     2221 tgcactgtaa attaccacat cagggtctcc ggggtcaacg ctgctaagca atggactgga
     2281 aaatttgtga cattgagttt ttggaataac tgggaatgct ggataactct ttgggtaaag
     2341 ctatggaaca aaagtaccag catgaactaa gaaatgactg tcacagcaca acatcaaaca
     2401 caatgcaaga caaaggttct ctgcttttgc cttacttgac agaaaaagta agctctaggt
     2461 tcatcgcatg cagacagcag caatacatga catatataaa ggctggctct tttctccctt
     2521 ccattgaact gaaacttcca ctcagtcaga cttgcggggg atccacagcc tgactaactc
     2581 gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt ttcagctcgc
     2641 atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac tgcagattgc
     2701 agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt tgacttccct
     2761 gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc cggaattcac
     2821 ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tggtgagcaa gggcgaggag
     2881 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2941 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3001 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3061 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3121 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3181 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3241 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3301 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3361 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3421 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     3481 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3541 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctgtgcc ttctagttgc
     3601 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3661 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3721 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3781 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa agggcgaatt
     3841 aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3901 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3961 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     4021 aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac cacaactaga
     4081 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4141 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4201 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattca
     4261 gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac gttgtctagc
     4321 aaggaagtga agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc
     4381 aggtggtaat gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt
     4441 ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg
     4501 agaccaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat
     4561 aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa
     4621 aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt
     4681 acagtaatca agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga
     4741 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     4801 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     4861 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     4921 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     4981 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5041 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     5101 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5161 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5221 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5281 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5341 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5401 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     5461 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     5521 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5581 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg
     5641 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5701 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5761 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5821 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5881 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5941 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6001 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6061 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6121 agcgtgagct atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg
     6181 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6241 atctttatag tcctgt
//
