LOCUS       sequence-329625-        6944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eCFP-PTEN(M265-N329)-mVenus in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6944)
  AUTHORS   Blum TR, Liu H, Packer MS, Xiong X, Lee PG, Zhang S, Richter M, 
            Minasov G, Satchell KJF, Dong M, Liu DR
  TITLE     Phage-assisted evolution of botulinum neurotoxin proteases with 
            reprogrammed specificity.
  JOURNAL   Science. 2021 Feb 19;371(6531):803-810. doi: 
            10.1126/science.abf5972.
  PUBMED    33602850
REFERENCE   2  (bases 1 to 6944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2021-02-19- 19"; volume: "371"; 
            issue: "6531"; pages: "803-810"
COMMENT     Sequence Label: pET-CVm-PTEN
FEATURES             Location/Qualifiers
     source          1..6944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(158..868,869..871,872..874))
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(200..221)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     549..568
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     808..829
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(join(881..895,896..904))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(1847..1876)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     RBS             1885..1907
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1922..1946
                     /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     complement(1946..1965)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1947..1965)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2049..2068)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2274..2351
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2352..3434
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2371..2390)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4243..4434
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4428..4450)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4536..4675
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4591..4610
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4690..4707)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4861..5449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4941..4960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5560..5579)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5571..6386
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5571..6386
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5644..5663)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6478..6933)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6615..6636)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6827..6846
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctt gtacagctcg tccatgccga
      181 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
      241 ggtctttgct cagggcggac tggtagctca ggtagtggtt gtcgggcagc agcacggggc
      301 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgccg ccgtcctcga
      361 tgttgtggcg gatcttgaag ttggccttga tgccgttctt ctgcttgtcg gcggtgatat
      421 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
      481 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
      541 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
      601 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga
      661 agcactgcag gccgtagccc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
      721 cggtggtgca gatcagcttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
      781 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
      841 ccccggtgaa cagctcctcg cccttgctca ccatcggatc cttatcgtca tcgtcgtaca
      901 gatcacttcc accactattt gaattcgctt tgtccagatc attcttagtc agcgtaagca
      961 ccaaatactc cttatcattg tcggcacgct cgatcgagca aatagagtca atctcctggt
     1021 cgcacaaaga gccgttttct actttttcag aagtctcttc aggcccaggg atgaagaacg
     1081 tgtttaccca gaaatggaac attttgtctt ttttcaaacg actacgccca cctttataca
     1141 gttcatccat accatgagta ataccagcgg cggtaacgaa ctctaaaagg accatatggt
     1201 cgcgtttctc attggggtcc ttagacaggg ctgactgtgt gctcaaatag tgattatcag
     1261 gtaaaaggac ggggccatct ccaattggtg tgttttgctg gtaatggtct gcaagctgga
     1321 cgcttccatc ctcaatgttg tgacgaatct tgaaatttgc cttgattccg tttttttgtt
     1381 tatcagccgt gatgtagacg ttatgagaga tatagttgta ttctaattta tgtcctaaga
     1441 tgttgccatc ctctttaaag tcgataccct tcaactcaat gcgattaaca agagtatctc
     1501 cctcaaactt aacctcggcg cgggtcttat agtttccatc atctttaaaa aaaatggtgc
     1561 gttcctggac gtaaccttct ggcatcgcgg acttgaaaaa gtcgtgttgt ttcatatggt
     1621 cagggtagcg agcgaaacac tgcacgcccc aggcgaacgt agtaaccaat gtaggccacg
     1681 gtactggaag cttgccggta gtgcaaataa attttaatgt caactttccg tacgtcgcat
     1741 cgccctctcc ttccccagat acggaaaact tgtgaccatt cacatcgcca tccagttcta
     1801 caagaatggg gaccacgccg gtaaacaatt cctccccctt agacatcaag tcttcctcag
     1861 agataagttt ttgttccatg tatatctcct tcttaaagtt aaacaaaatt atttctagag
     1921 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     1981 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     2041 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     2101 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     2161 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     2221 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     2281 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     2341 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     2401 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     2461 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     2521 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     2581 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     2641 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     2701 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     2761 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     2821 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     2881 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     2941 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     3001 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     3061 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     3121 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     3181 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     3241 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     3301 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     3361 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3421 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     3481 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     3541 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     3601 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     3661 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     3721 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     3781 gtgcgcatga tcgtgctcct gtcgttgagg actcggctag gctggcgggg ttgccttact
     3841 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaact
     3901 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3961 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     4021 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     4081 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     4141 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     4201 attaccccca tgaacagaaa tcccccttac acgtaggcat cagtgaccaa acaggaaaaa
     4261 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     4321 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     4381 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4441 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4501 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     4561 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4621 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     4681 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4741 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4801 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4861 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4981 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5041 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5101 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5161 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5221 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5281 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5341 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5461 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5521 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     5581 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     5641 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     5701 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     5761 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     5821 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     5881 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     5941 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     6001 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     6061 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     6121 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     6181 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     6241 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     6301 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     6361 atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt
     6421 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6481 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6541 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6601 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6661 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6721 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa tggagccccc
     6781 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6841 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6901 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
