LOCUS       Exported                5750 bp ds-DNA     circular SYN 16-MAR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5750)
  AUTHORS   SnapGene License
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Mar 20, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5750
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1493..2463
                     /organism="Homo sapiens"
                     /chromosome="20"
                     /map="20q11.23"
                     /mol_type="mRNA"
                     /db_xref="taxon:9606"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     primer_bind     141..190
                     /label=F-D1
     primer_bind     147..194
                     /label=F-Mlu-D2
     primer_bind     147..161
                     /label=3'-pcalm-ampF
     primer_bind     150..168
                     /label=P-Calm1-F
     primer_bind     151..200
                     /label=F-D2
     misc_feature    156..276
                     /label=P Calm1
     misc_feature    156..165
                     /label=D1
     primer_bind     161..210
                     /label=F-D3
     primer_bind     complement(161..185)
                     /label=R-D4
     misc_feature    166..175
                     /label=D2
     primer_bind     171..220
                     /label=F-D4
     primer_bind     complement(171..195)
                     /label=R-D5
     misc_feature    176..185
                     /label=D3
     primer_bind     181..230
                     /label=F-D5
     primer_bind     complement(181..205)
                     /label=R-D6
     misc_feature    186..195
                     /label=D4
     primer_bind     191..240
                     /label=F-D6
     primer_bind     complement(191..215)
                     /label=r-d7
     misc_feature    196..205
                     /label=D5
     primer_bind     200..250
                     /label=F-D7
     primer_bind     complement(201..225)
                     /label=R-D8
     misc_feature    206..215
                     /label=D6
     primer_bind     211..260
                     /label=F-D8
     primer_bind     complement(215..236)
                     /label=R-D9
     misc_feature    216..225
                     /label=D7
     primer_bind     222..271
                     /label=F-D9
     primer_bind     complement(222..246)
                     /label=R-D10
     misc_feature    226..235
                     /label=D8
     primer_bind     complement(232..256)
                     /label=R-D11
     misc_feature    236
                     /label=+1
     misc_feature    237..246
                     /label=D9
     primer_bind     complement(242..266)
                     /label=R-D12
     misc_feature    247..256
                     /label=D10
     misc_feature    257..266
                     /label=D11
     primer_bind     complement(260..287)
                     /label=2B-1R
     primer_bind     complement(261..276)
                     /label=P-Calm1-R
     misc_feature    267..276
                     /label=D12
     primer_bind     271..296
                     /label=2B-2F
     primer_bind     complement(277..298)
                     /label=Primer 1
     misc_feature    277..282
                     /label=BsiWI
     primer_bind     283..302
                     /label=Primer 5
     CDS             288..1318
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     misc_feature    548
                     /label=silent mut
     primer_bind     complement(616..650)
                     /label=R-Cre-R119STOP
     primer_bind     complement(632..651)
                     /label=R-CRE-L115-BamHI
     primer_bind     636..670
                     /label=F-Cre-R119STOP
     primer_bind     1093..1116
                     /label=F-Seq-Cre-3F1
     misc_feature    1093..1098
                     /label=ClaI
     misc_feature    1224
                     /label=mut
     primer_bind     1262..1279
                     /label=cre-SF
     primer_bind     complement(1296..1318)
                     /label=R-T5-Cre
     intron          1320..1360
                     /label=Splicing donor
                     /note="Splicing donor"
     primer_bind     complement(1338..1367)
                     /label=2B-2R
     primer_bind     complement(1341..1385)
                     /label=Primer 7
     primer_bind     complement(1341..1385)
                     /label=Primer 6
     primer_bind     1350..1387
                     /label=2B-3F
     primer_bind     complement(1356..1380)
                     /label=Primer 3
     primer_bind     1367..1401
                     /label=Primer 8
     primer_bind     1367..1401
                     /label=Primer 2
     primer_bind     1367..1401
                     /label=F-T5-JT+2272
     protein_bind    1367..1400
                     /label=loxJTZ17?2272?
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    1380..1387
                     /label=2272
     intron          1401..1492
                     /label=Splicing acceptor
                     /note="Splicing acceptor"
     primer_bind     1401..1420
                     /label=Primer 4
     primer_bind     1459..1478
                     /label=T0060_PIRES-F
     primer_bind     complement(1468..1492)
                     /label=#57-R-SA-3R1
     primer_bind     1477..1512
                     /label=A12F-NheI-F
     primer_bind     1477..1492
                     /label=Primer 10
     misc_feature    1492
                     /label=NheI?
     CDS             1493..2463
                     /codon_start=1
                     /gene="RALGAPB"
                     /gene_synonym="KIAA1219; RalGAPbeta"
                     /product="ral GTPase-activating protein subunit beta
                     isoform 1"
                     /label=RALGAPB
                     /note="isoform 1 is encoded by transcript variant 1; Ral
                     GTPase activating protein, beta subunit (non-catalytic); 
                     Ral GTPase activating protein non-catalytic beta subunit"
                     /db_xref="GI:34787409"
                     /db_xref="CCDS:CCDS13305.1"
                     /db_xref="GeneID:57148"
                     /db_xref="HGNC:HGNC:29221"
                     /db_xref="MIM:618833"
                     /protein_id="NP_065069.1"
                     /translation="QKTNQEILKNVESSRTVQPHFLEFLLSLGWSVDVGRHPGWTGHVS
                     TSWSINCCDDGEGSQQEVISSEDIGASIFNGQKKVLYYADALTEIAFVVPSPVESLTDS
                     LESNISDQDSDSNMDLMPGILKQPSLTLELFPNHTDNLNSSQRLSPSSRMRKLPQGRPV
                     PPLGPETRVSVVWVERYDDIENFPLSELMTEISTGVETTANSSTSLRSTTLEKEVPVIF
                     IHPLNTGLFRIKIQGATGKFNMVIPLVDGMIVSRRALGFLVRQTVINICRRKRLESDSY
                     SPPHVRRKQKITDIVNKYRNKQLEPEFYTSLFQEVGLKNCSS"
     exon            1493..1512
                     /gene="RALGAPB"
                     /gene_synonym="KIAA1219; RalGAPbeta"
                     /inference="alignment:same species:2.1.0"
     misc_feature    1493..1494
                     /label=Feature 49
     primer_bind     complement(2443..2484)
                     /label=A12F-BsrGI-R
     misc_feature    2464..2469
                     /label=BsrGI
     CDS             2470..2535
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2475..2563)
                     /label=296
     terminator      2539..2970
                     /label=W3SL
                     /note="Short WPRE and SV40 polyA"
     primer_bind     2539..2564
                     /label=#56-F-W3SL-5F1
     primer_bind     complement(2618..2641)
                     /label=R-Seq-W3SL-5R1
     misc_feature    3013..3153
                     /label=R-ITR
     rep_origin      3156..3611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3303..3312)
                     /label=F-Seq-Cre-3F1
     promoter        3893..3997
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3998..4858
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="HSNMYPLMRQ*P**MLQ*Y*KRKSMSIQHFRVALIPFFAAFCLPV
                     FAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLS
                     RIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTT
                     IGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLT
                     LASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIV
                     VIYTTG"
     rep_origin      5029..5617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtcgctc tgcgcgaggc ggctccgccg
      181 cggcggaggg atacggcgca ccatatatat atcgcggggc gcagactcgc gctccggcag
      241 tggtgctggg agtgtcgtgg acgccgtgcc gttactcgta cgccaccatg tccaatttac
      301 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
      361 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
      421 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
      481 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
      541 taaaaaccat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
      601 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
      661 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
      721 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
      781 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
      841 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
      901 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
      961 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     1021 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     1081 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     1141 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     1201 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgta atgaactata
     1261 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagg
     1321 taagtatcaa ggttacaaga caggtttaag gagaccaata actagtataa cttcgtatag
     1381 gatactttat agcaatttat gaaactgggc ttgtcgagac agagaagact cttgcgtttc
     1441 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggtcagaaa
     1501 acgaaccaag agattttaaa gaatgtggag tcttccagaa ctgttcagcc acatttccta
     1561 gaatttttgc tttcccttgg ctggtcagta gatgtgggca gacaccctgg ttggactggg
     1621 catgtttcta ccagttggtc tattaattgt tgtgatgatg gtgaaggatc tcaacaagaa
     1681 gtgatttcct ctgaagatat tggagctagc attttcaatg gacagaagaa ggtgctgtat
     1741 tatgctgatg cccttacaga aattgctttt gtggttcctt ctcctgtgga gtccttaact
     1801 gattcattgg aaagtaacat ctcggaccaa gatagtgatt caaatatgga tcttatgcca
     1861 ggaattctga aacagccatc cctgacactt gagcttttcc ccaatcatac agacaatctt
     1921 aattcctcac agaggctcag tcccagttcc agaatgagga agctgcctca gggtcgccct
     1981 gttcctcccc ttggacctga gacaagagtt tctgtagtct gggtggaacg ctatgatgat
     2041 atagaaaact ttcccctctc agagctgatg acagagatca gtactggtgt ggaaactact
     2101 gcaaatagta gcacttcact gagatctaca actcttgaaa aagaagttcc tgtcatcttc
     2161 atccaccctt taaacactgg attattccgg ataaaaattc aaggagccac tggaaaattt
     2221 aatatggtca tccctcttgt ggatgggatg attgtcagca ggcgagctct tggctttctg
     2281 gtgaggcaga ctgtaattaa catttgtaga agaaagagac tggaaagtga ctcctacagt
     2341 cccccccatg tccgccggaa acagaaaatc accgacattg tcaacaagta ccggaacaag
     2401 cagctggagc cagagtttta tacttcactt ttccaggagg ttggactcaa gaactgcagt
     2461 tcttgtacag actacaaaga ccatgacggt gattataaag atcatgacat cgactacaag
     2521 gatgacgatg acaagtaact cgagataatc aacctctgga ttacaaaatt tgtgaaagat
     2581 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     2641 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     2701 ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     2761 ctcggctgtt gggcactgac aattccgtgg tgtttatttg tgaaatttgt gatgctattg
     2821 ctttatttgt aaccatctag ctttatttgt gaaatttgtg atgctattgc tttatttgta
     2881 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     2941 gttcaggggg agatgtggga ggttttttaa agatctggag accggtgtgg cacgtgcgga
     3001 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3061 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     3121 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     3181 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     3241 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3301 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3361 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3421 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     3481 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3541 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     3601 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3661 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     3721 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3781 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3841 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     3901 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     3961 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4021 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4081 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4141 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4201 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4261 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4321 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4381 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4441 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4501 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4561 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4621 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4681 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4741 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4801 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4861 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4921 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4981 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5041 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5101 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5161 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5221 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5281 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5341 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5401 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5461 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5521 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5581 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5641 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5701 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
