LOCUS       sequence-390121-        8768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mini-Agrin in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8768)
  TITLE     Jeff Miner Lab Plasmids
REFERENCE   2  (bases 1 to 8768)
  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"
COMMENT     Sequence Label: pCI_E01_mini-mAgrin-Myc-His-WPRE__LG2
FEATURES             Location/Qualifiers
     source          1..8768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    39..322
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        513..892
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        893..1096
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1047..1067
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1232..1364
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1347..1366
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1408..1427
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1408..1426
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1527..1536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3918..3938
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3963..3992
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4002..4031
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4041..4070
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4080..4109
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4119..4148
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4176..4193
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4230..4818
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4283..4303)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4701..4712)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4906..4924)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    4961..5082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4972..4991)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5026..5045
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5327..5684
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5327..5518
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5350..5370)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5535..5670
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5715..6752
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             6731..6739
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    6776..6824
                     /note="synthetic polyadenylation signal"
     promoter        6980..7084
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7085..7153,7154..7945)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7303..7322)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(8103..8691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8183..8202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attatcaaaa aggatcttca cctagatcct tttatagtcc ggaaatacag gaacgcacgc
       61 tggatggccc ttcgctggga tggtgaaacc atgaaaaatg gcagcttcag tggattaagt
      121 gggggtaatg tggcctgtac cctctggttg cataggtatt catacggtta aaatttatca
      181 ggcgcgattg cggcagtttt tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg
      241 atcgcgctga acgcgtttta gcggtgcgta caattaaggg attatggtaa atccacttac
      301 tgtctgccct cgtagccatc gagataaacc gcagtactcc ggccacgatg cgtccggcgt
      361 agaggatcga gatcttcaat attggccatt agccatatta ttcattggtt atatagcata
      421 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
      481 tattggctca tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata
      541 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      601 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      661 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      721 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
      781 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
      841 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      901 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      961 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1021 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1081 ctatataagc agagctggtt tagtgaaccg tcagatcact agaagcttta ttgcggtagt
     1141 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
     1201 ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta
     1261 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     1321 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt
     1381 caattacagc tcttaaggct agagtattaa tacgactcac tatagggcta gcaaagcgat
     1441 cgccgaactc acctatctcc ccaacaccta ataacattca atcactcttt ccactaacca
     1501 cctatctaca tcaccaccca ccagccgcca ccatggtccg cccgcggctg agcttcccgg
     1561 ccccgctgct gccgctgctg ctgctcctgg cggcggccgc gccagccgtg ccgggcgcca
     1621 gcgggacgtg cccggagcgc gccttggagc ggcgcgagga ggaggcgaac gtggtgctca
     1681 ccggcacagt ggaggagatc ctcaacgtgg acccggtgca gcacacctac tcctgcaagg
     1741 ttcgagtgtg gcggtacttg aaaggcaaag atgtggtggc ccaagagagc ctgctggatg
     1801 gtggcaacaa ggtggtaatt ggtggctttg gagaccccct catctgcgac aaccaggtgt
     1861 ccaccgggga cactagaatc ttctttgtga acccagcacc accctacctg tggccagccc
     1921 acaagaatga gttgatgctc aactccagcc ttatgcggat cactttgcgg aacctggaag
     1981 aggtggagtt ctgtgtagaa gacaaacctg ggatccactt cacagcagcg ccttctatgc
     2041 ctcctgatgt gtgcagggga atgttatgtg gctttggtgc tgtgtgtgaa cctagtgttg
     2101 aggatccagg ccgggcctcc tgtgtgtgca agaagaatgt ctgccctgct atggtagctc
     2161 ctgtgtgtgg ctcagatgct tccacctata gcaacgagtg tgagctacag cgtgcacagt
     2221 gcaaccagca acggcgcatc cgcctactcc gccaagggcc atgtgggtcc cgggacccct
     2281 gtgccaatgt gaccgctagc actccaggcc cgcaacggcc cccaaagtcc tgtgattccc
     2341 agccttgcct ccacggaggt acctgccagg acctggattc tggcaagggt ttcagctgca
     2401 gctgtactgc aggcagggct ggcactgtct gtgagaaagt gcaactcccc tctgtgccag
     2461 cttttaaggg ccactccttc ttggccttcc ccaccctccg agcctaccac acgctgcgtc
     2521 tggcactaga attccgggcg ctggagacag agggactgct gctctacaat ggcaatgcac
     2581 gtggcaaaga tttcctggct ctggctctgt tggatggtca tgtacagttc aggttcgaca
     2641 cgggctcagg gccggcggtg ctaacaagct tagtgccagt ggaaccggga cggtggcacc
     2701 gcctcgagtt gtcacggcat tggcggcagg gcacactttc tgtggatggc gaggctcctg
     2761 ttgtaggtga aagtcccagt gggactgatg gcctcaactt ggacacgaag ctctatgtgg
     2821 gtggtctccc agaagaacaa gttgccacgg tgcttgatcg gacctctgtg ggcatcggcc
     2881 tgaaaggatg cattcgtatg ttggacatca acaaccagca gctggagctg agcgattggc
     2941 agagggctgt ggttcaaagc tctggtgtgg gggaatgcgg agaccatccc tgctcaccta
     3001 acccctgcca tggcggggcc ctctgccagg ccctggaggc tggcgtgttc ctctgtcagt
     3061 gtccacctgg ccgctttggc ccaacttgtg cagatgaaaa gaacccctgc caaccgaacc
     3121 cctgccacgg gtcagccccc tgccatgtgc tttccagggg tggggccaag tgtgtgtgcc
     3181 ccctgggacg cagtggttcc ttctgtgaga cagtcctgga gaatgctggc tcccggaccc
     3241 aggccgtgga caacccctgc cttaatgggg gctcctgtat cccgagggaa gccacttatg
     3301 agtgcctgtg tcctgggggc ttctctgggc tgcactgcga gaaggggata gttgagaagt
     3361 cagtggggga cctagaaaca ctggcctttg atgggcggac ctacatcgag tacctcaatg
     3421 ctgtgactga gagtgagaaa gcgctgcaga gcaaccactt tgagctgagc ttacgcactg
     3481 aggccacgca ggggctggtg ctgtggattg gaaaggttgg agaacgtgca gactacatgg
     3541 ctctggccat tgtggatggg cacctacaac tgagctatga cctaggctcc cagccagttg
     3601 tgctgcgctc cactgtgaag gtcaacacca accgctggct tcgagtcagg gctcacaggg
     3661 agcacaggga aggttccctt caggtgggca atgaagcccc tgtgactggc tcttccccgc
     3721 tgggtgccac acaattggac acagatggag ccctgtggct tggaggccta cagaagcttc
     3781 ctgtggggca ggctctcccc aaggcctatg gcacgggttt tgtgggctgt ctgcgggacg
     3841 tggtagtggg ccatcgccag ctgcatctgc tggaggacgc tgtcaccaaa ccagagctaa
     3901 gaccctgccc cactctcgag gatctgtact ttcagagcat cgatggaagt ggtaaagcta
     3961 tggagcaaaa gctcatttct gaagaggact tgaatgaaat ggagcaaaag ctcatttctg
     4021 aagaggactt gaatgaaatg gagcaaaagc tcatttctga agaggacttg aatgaaatgg
     4081 agcaaaagct catttctgaa gaggacttga atgaaatgga gcaaaagctc atttctgaag
     4141 aggacttgaa tgaaatggag agcttgggcg acctccatca tcaccatcac cactagggga
     4201 tatcagctga attctagaaa ttcatcgaca atcaacctct ggattacaaa atttgtgaaa
     4261 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4321 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4381 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4441 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4501 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4561 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4621 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4681 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4741 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4801 tttgggccgc ctccccgccc aggttccttg ggccgcgact ctagggcatg caagctgatc
     4861 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     4921 tagcataacc ccttggggcg gccgcttcga gcagacatga taagatacat tgatgagttt
     4981 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     5041 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     5101 cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa gtaaaacctc
     5161 tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt cctgatgcgg
     5221 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac
     5281 catggcctga aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga
     5341 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5401 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5461 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5521 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5581 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     5641 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc gattcttctg
     5701 acactagcgc caccatgaag aagcccgaac tcaccgctac cagcgttgaa aaatttctca
     5761 tcgagaagtt cgacagtgtg agcgacctga tgcagttgtc ggagggcgaa gagagccgag
     5821 ccttcagctt cgatgtcggc ggacgcggct atgtactgcg ggtgaatagc tgcgctgatg
     5881 gcttctacaa agaccgctac gtgtaccgcc acttcgccag cgctgcacta cccatccccg
     5941 aagtgttgga catcggcgag ttcagcgaga gcctgacata ctgcatcagt agacgcgccc
     6001 aaggcgttac tctccaagac ctccccgaaa cagagctgcc tgctgtgtta cagcctgtcg
     6061 ccgaagctat ggatgctatt gccgccgccg acctcagtca aaccagcggc ttcggcccat
     6121 tcgggcccca aggcatcggc cagtacacaa cctggcggga tttcatttgc gccattgctg
     6181 atccccatgt ctaccactgg cagaccgtga tggacgacac cgtgtccgcc agcgtagctc
     6241 aagccctgga cgaactgatg ctgtgggccg aagactgtcc cgaggtgcgc cacctcgtcc
     6301 atgccgactt cggcagcaac aacgtcctga ccgacaacgg ccgcatcacc gccgtaatcg
     6361 actggtccga agctatgttc ggggacagtc agtacgaggt ggccaacatc ttcttctggc
     6421 ggccctggct ggcttgcatg gagcagcaga ctcgctactt cgagcgccgg catcccgagc
     6481 tggccggcag ccctcgtctg cgagcctaca tgctgcgcat cggcctggat cagctctacc
     6541 agagcctcgt ggacggcaac ttcgacgatg ctgcctgggc tcaaggccgc tgcgatgcca
     6601 tcgtccgcag cggggccggc accgtcggtc gcacacaaat cgctcgccgg agcgcagccg
     6661 tatggaccga cggctgcgtc gaggtgctgg ccgacagcgg caaccgccgg cccagtacac
     6721 gaccgcgcgc taaggaggta ggtcgagttt agactctagc catcacgatg gccgcaataa
     6781 aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag
     6841 gatcctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
     6901 gggtcagcac cgtttctgcg gactggcttt ctacgtaatg gtttcttaga cgtcaggtgg
     6961 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     7021 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     7081 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     7141 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     7201 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     7261 ccccgaagaa cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg gcgcggtatt
     7321 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     7381 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     7441 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     7501 tatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     7561 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7621 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7681 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7741 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7801 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7861 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7921 tgcctcactg attaagcatt ggtaattcga aatgaccgac caagcgacgc ccaaccggta
     7981 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     8041 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     8101 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     8161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     8221 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     8281 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     8341 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     8401 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     8461 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     8521 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     8581 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     8641 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct
     8701 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     8761 gtcatgag
//
