LOCUS       Exported                6767 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 6767)
  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..6767
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1493..3480
                     /organism="Homo sapiens"
                     /chromosome="3"
                     /map="3p12.3"
                     /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=B11F-NheI-F
     primer_bind     1477..1492
                     /label=Primer 10
     misc_feature    1492
                     /label=NheI?
     CDS             1493..3480
                     /codon_start=1
                     /gene="ROBO2"
                     /gene_synonym="SAX3"
                     /product="roundabout homolog 2 isoform 23 precursor"
                     /label=ROBO2
                     /note="isoform 23 precursor is encoded by transcript
                     variant 23; roundabout, axon guidance receptor, homolog 2"
                     /db_xref="GI:2074014171"
                     /db_xref="CCDS:CCDS93319.1"
                     /db_xref="GeneID:6092"
                     /db_xref="HGNC:HGNC:10250"
                     /db_xref="MIM:602431"
                     /protein_id="NP_001382585.1"
                     /translation="IQYRVEVAASTSAGVGVKSEPQPIIIGRRNEVVITENNNSITEQI
                     TDVVKQPAFIAGIGGACWVILMGFSIWLYWRRKKRKGLSNYAVTFQRGDGGLMSNGSRP
                     GLLNAGDPSYPWLADSWPATSLPVNNSNSGPNEIGNFGRGDVLPPVPGQGDKTATMLSD
                     GAIYSSIDFTTKTSYNSSSQITQATPYATTQILHSNSIHELAVDLPDPQWKSSIQQKTD
                     LMGFGYSLPDQNKGNNGGKGGKKKKNKNSSKPQKNNGSTWANVPLPPPPVQPLPGTELE
                     HYAVEQQENGYDSDSWCPPLPVQTYLHQGLEDELEEDDDRVPTPPVRGVASSPAISFGQ
                     QSTATLTPSPREEMQPMLQAHLDELTRAYQFDIAKQTWHIQSNNQPPQPPVPPLGYVSG
                     ALISDLETDVADDDADDEEEALEIPRPLRALDQTPGSSMDNLDSSVTGKAFTSSQRPRP
                     TSPFSTDSNTSAALSQSQRPRPTKKHKGGRMDQQPALPHRREGMTDDLPPPPDPPPGQG
                     LRQQIGPSQQAGNVENSAERKGSSLERQHASSLEDTKSSLDCPARTSLEWQRQTQEWIS
                     STERQEDIRKAPHKQGVGSEEALVPYSKPSFPSPGGHSSSGTASSKGSTGPRKTEVLRA
                     GHQRNASDLLDIGYMGSNSQGQFTGEL"
     misc_feature    1493..1494
                     /label=Feature 50
     primer_bind     2039..2055
                     /label=B11B-ROBO2-SF
     primer_bind     2086..2102
                     /label=B11F-SF
     primer_bind     complement(3460..3501)
                     /label=B11F-BsrGI-R
     misc_feature    3481..3486
                     /label=BsrGI
     CDS             3487..3552
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3492..3580)
                     /label=296
     terminator      3556..3987
                     /label=W3SL
                     /note="Short WPRE and SV40 polyA"
     primer_bind     3556..3581
                     /label=#56-F-W3SL-5F1
     primer_bind     complement(3635..3658)
                     /label=R-Seq-W3SL-5R1
     misc_feature    4030..4170
                     /label=R-ITR
     rep_origin      4173..4628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4320..4329)
                     /label=F-Seq-Cre-3F1
     promoter        4910..5014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5015..5875
                     /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      6046..6634
                     /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 aggtattcaa
     1501 taccgggtag aggttgcagc tagtaccagt gcaggggttg gagtaaagag tgagccacag
     1561 ccaataataa tcgggagacg caatgaagtt gtcattactg aaaacaataa cagcataact
     1621 gagcaaatca ctgatgtggt gaagcaacca gcctttatag ctggtattgg tggtgcctgc
     1681 tgggtaattc tgatgggttt tagcatatgg ttgtattggc gaagaaagaa gaggaaggga
     1741 ctcagtaatt atgctgttac gtttcaaaga ggagatggag gactaatgag caatggaagc
     1801 cgtccaggtc ttctcaatgc tggtgatccc agctatccat ggcttgctga ttcttggcca
     1861 gccacgagct tgccagtaaa taatagcaac agtggcccaa atgagattgg aaattttggc
     1921 cgtggagatg tgctgccacc agttccaggc caaggggata aaacagcaac gatgctctca
     1981 gatggagcca tttatagtag cattgacttc actaccaaaa ccagttacaa cagttccagc
     2041 caaataacac aggctacccc atatgccacg acacagatct tgcattccaa cagcatacat
     2101 gaattggctg tcgatctgcc tgatccacaa tggaaaagct caattcagca aaaaacagat
     2161 ctgatgggat ttggttattc tctacctgat cagaacaaag gtaacaatgg tgggaaaggt
     2221 ggaaaaaaga agaaaaataa aaactcttct aaaccacaga aaaacaatgg atccacttgg
     2281 gccaatgtcc ctctacctcc ccccccagtc cagccccttc ctggcacgga gctggaacac
     2341 tatgcagtgg aacaacaaga aaatggctat gacagtgata gctggtgccc accattgcca
     2401 gtacaaactt acttacacca aggtctggaa gatgaactgg aagaagatga tgatagggtc
     2461 ccaacacctc ctgttcgagg cgtggcttct tctcctgcta tctcctttgg acagcagtcc
     2521 actgcaactc ttactccatc cccacgggaa gagatgcaac ccatgctgca ggctcacctg
     2581 gatgagttga caagagccta tcagtttgat atagcaaaac aaacatggca cattcaaagc
     2641 aataatcaac ctccacagcc tccagttcca ccgttaggtt atgtgtctgg agccttgatt
     2701 tctgatttgg aaacggatgt tgcagatgat gatgccgacg acgaagagga agctttagaa
     2761 atccccaggc ccctgagagc actggaccag actcctggat ccagcatgga caatctagac
     2821 agctctgtga caggaaaagc ctttacctcc tctcaaagac ctcgacctac cagcccattt
     2881 tctactgaca gtaacaccag tgcagccctg agtcaaagtc agaggcctcg gcccactaaa
     2941 aaacacaagg gagggcggat ggaccaacaa ccagcattgc ctcatcgaag ggaaggaatg
     3001 acagatgatc ttccaccacc accagatccc ccgccaggtc agggtttaag gcagcaaata
     3061 ggcccgagcc agcaggctgg taacgtggaa aactcagcag agagaaaagg aagctctcta
     3121 gagagacaac atgcatccag cttagaagac acaaagagct cattggattg tccagctaga
     3181 acctccctag agtggcagcg acaaacccag gaatggataa gctccacaga acgacaagaa
     3241 gatatacgga aagccccaca caaacaaggt gtcggatcag aggaggcctt ggtgccctat
     3301 agcaagccca gtttcccatc tccaggtggc cacagctcat caggaacagc ttcttctaag
     3361 ggatccactg gacctaggaa aaccgaggtg ttgagagcag gccaccagcg caatgccagc
     3421 gaccttcttg acataggata tatgggctcc aacagtcaag gacagtttac aggtgaatta
     3481 tgtacagact acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat
     3541 gacgatgaca agtaactcga gataatcaac ctctggatta caaaatttgt gaaagattga
     3601 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3661 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3721 tagttcttgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3781 ggctgttggg cactgacaat tccgtggtgt ttatttgtga aatttgtgat gctattgctt
     3841 tatttgtaac catctagctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3901 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3961 cagggggaga tgtgggaggt tttttaaaga tctggagacc ggtgtggcac gtgcggaccg
     4021 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4081 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4141 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4201 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4261 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4321 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4381 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4441 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4501 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4561 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4621 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4681 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4741 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4801 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4861 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4921 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4981 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5041 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5101 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5161 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5221 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5281 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5341 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5401 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5461 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5521 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5581 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5641 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5701 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5761 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5821 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5881 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5941 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6001 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6061 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6121 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6181 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6241 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6301 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6361 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6421 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6481 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6541 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6601 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6661 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6721 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
