LOCUS       pMIA4.721_Ts18d_        7355 bp DNA     circular SYN 05-JAN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7355)
  AUTHORS   Matias
  TITLE     Direct Submission
  JOURNAL   Exported Jan 5, 2024 from SnapGene 7.1.0
            https://www.snapgene.com
COMMENT     Sequence Label: pMIA4.721-Olonne
FEATURES             Location/Qualifiers
     source          1..7355
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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"
     rep_origin      1206..1879
                     /label=pUC
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2010..2050
                     /label=Ts18 5' into  4.721_fwd
     variation       2048
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs1022352845"
     variation       2049
                     /replace="G/A/T"
                     /db_xref="dbSNP:rs906520158"
     variation       2052
                     /replace="-/TGC"
                     /db_xref="dbSNP:rs1164962202"
     variation       2052
                     /replace="A/G"
                     /db_xref="dbSNP:rs979408837"
     variation       2053
                     /replace="A/C"
                     /db_xref="dbSNP:rs1384525476"
     variation       2054
                     /replace="T/-"
                     /db_xref="dbSNP:rs1179239292"
     variation       2055..2057
                     /replace="AAA/A"
                     /db_xref="dbSNP:rs1471025503"
     variation       2055
                     /replace="A/G"
                     /db_xref="dbSNP:rs1231000999"
     variation       2057
                     /replace="A/C"
                     /db_xref="dbSNP:rs1202642820"
     variation       2060
                     /replace="A/G"
                     /db_xref="dbSNP:rs1437486471"
     variation       2061
                     /replace="T/C"
                     /db_xref="dbSNP:rs746854756"
     variation       2062
                     /replace="C/G"
                     /db_xref="dbSNP:rs1292516825"
     variation       2065
                     /replace="A/C"
                     /db_xref="dbSNP:rs1598896204"
     variation       2067
                     /replace="T/C"
                     /db_xref="dbSNP:rs1598896206"
     variation       2069
                     /replace="A/C"
                     /db_xref="dbSNP:rs1598896207"
     variation       2071
                     /replace="C/T"
                     /db_xref="dbSNP:rs1224822112"
     variation       2082
                     /replace="A/G"
                     /db_xref="dbSNP:rs927506881"
     variation       2083
                     /replace="T/C"
                     /db_xref="dbSNP:rs1419071305"
     variation       2092
                     /replace="T/C"
                     /db_xref="dbSNP:rs1296079292"
     variation       2093
                     /replace="A/G"
                     /db_xref="dbSNP:rs1598896209"
     variation       2096
                     /replace="A/C"
                     /db_xref="dbSNP:rs1598896211"
     variation       2097
                     /replace="C/T"
                     /db_xref="dbSNP:rs1291675944"
     variation       2099
                     /replace="G/C"
                     /db_xref="dbSNP:rs1224876966"
     variation       2101
                     /replace="C/A"
                     /db_xref="dbSNP:rs958858505"
     variation       2104
                     /replace="T/C"
                     /db_xref="dbSNP:rs1278180821"
     variation       2109
                     /replace="A/G"
                     /db_xref="dbSNP:rs1440969043"
     variation       2116
                     /replace="C/G"
                     /db_xref="dbSNP:rs1034124262"
     variation       2119
                     /replace="T/C"
                     /db_xref="dbSNP:rs1351467332"
     variation       2123
                     /replace="C/T"
                     /db_xref="dbSNP:rs895222698"
     variation       2124
                     /replace="G/A"
                     /db_xref="dbSNP:rs1422197476"
     variation       2125..2134
                     /replace="CTTCTATCTT/CTTCTATCTTCTATCTT"
                     /db_xref="dbSNP:rs1364462315"
     variation       2130
                     /replace="A/G"
                     /db_xref="dbSNP:rs1006090846"
     variation       2138
                     /replace="T/C"
                     /db_xref="dbSNP:rs1016067203"
     variation       2139
                     /replace="C/A"
                     /db_xref="dbSNP:rs1402407159"
     variation       2145
                     /replace="G/A"
                     /db_xref="dbSNP:rs1405460339"
     variation       2153
                     /replace="C/A"
                     /db_xref="dbSNP:rs181485563"
     variation       2158
                     /replace="A/T"
                     /db_xref="dbSNP:rs1036378975"
     variation       2160
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs971946146"
     variation       2162
                     /replace="T/C"
                     /db_xref="dbSNP:rs147058900"
     variation       2168
                     /replace="C/A"
                     /db_xref="dbSNP:rs1026255628"
     variation       2184
                     /replace="C/A/G/T"
                     /db_xref="dbSNP:rs781050369"
     variation       2190
                     /replace="C/A"
                     /db_xref="dbSNP:rs930599926"
     variation       2197
                     /replace="T/C"
                     /db_xref="dbSNP:rs1319280712"
     variation       2200
                     /replace="T/C/G"
                     /db_xref="dbSNP:rs987143875"
     variation       2201
                     /replace="C/T"
                     /db_xref="dbSNP:rs1282528139"
     variation       2211
                     /replace="G/A"
                     /db_xref="dbSNP:rs9951713"
     variation       2212
                     /replace="C/T"
                     /db_xref="dbSNP:rs943092970"
     variation       2213
                     /replace="C/T"
                     /db_xref="dbSNP:rs1284777791"
     variation       2216
                     /replace="T/C"
                     /db_xref="dbSNP:rs1402760512"
     variation       2221
                     /replace="T/C"
                     /db_xref="dbSNP:rs888985250"
     variation       2223
                     /replace="C/G"
                     /db_xref="dbSNP:rs1298299621"
     variation       2225
                     /replace="C/G"
                     /db_xref="dbSNP:rs1432737828"
     variation       2229
                     /replace="T/C"
                     /db_xref="dbSNP:rs1343369719"
     variation       2231
                     /replace="T/C"
                     /db_xref="dbSNP:rs1355026500"
     variation       2240
                     /replace="T/C"
                     /db_xref="dbSNP:rs1568082857"
     variation       2242
                     /replace="G/A"
                     /db_xref="dbSNP:rs1482968945"
     variation       2251
                     /replace="C/T"
                     /db_xref="dbSNP:rs1414813898"
     variation       2260
                     /replace="A/G"
                     /db_xref="dbSNP:rs1240114796"
     variation       2261
                     /replace="T/C"
                     /db_xref="dbSNP:rs989641498"
     variation       2262
                     /replace="G/A"
                     /db_xref="dbSNP:rs1282592535"
     variation       2263
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs1453625740"
     variation       2266
                     /replace="C/T"
                     /db_xref="dbSNP:rs915379203"
     variation       2268
                     /replace="G/T"
                     /db_xref="dbSNP:rs1187203488"
     variation       2269
                     /replace="C/A"
                     /db_xref="dbSNP:rs1486926146"
     variation       2271
                     /replace="G/C"
                     /db_xref="dbSNP:rs1240846631"
     variation       2272
                     /replace="T/A"
                     /db_xref="dbSNP:rs1222777979"
     variation       2277
                     /replace="G/A"
                     /db_xref="dbSNP:rs1246303817"
     variation       2286..2290
                     /replace="AAAAA/AAA/AAAAAA"
                     /db_xref="dbSNP:rs1265420957"
     variation       2292
                     /replace="A/G"
                     /db_xref="dbSNP:rs1221252257"
     variation       2295
                     /replace="C/T"
                     /db_xref="dbSNP:rs545648099"
     variation       2296
                     /replace="G/A"
                     /db_xref="dbSNP:rs564007948"
     variation       2297
                     /replace="A/G"
                     /db_xref="dbSNP:rs762716904"
     variation       2303
                     /replace="T/C"
                     /db_xref="dbSNP:rs1568082870"
     variation       2305
                     /replace="T/G"
                     /db_xref="dbSNP:rs1438997992"
     variation       2310
                     /replace="T/A/G"
                     /db_xref="dbSNP:rs1156799038"
     variation       2311
                     /replace="C/T"
                     /db_xref="dbSNP:rs1288195317"
     variation       2318
                     /replace="A/T"
                     /db_xref="dbSNP:rs1453339726"
     variation       2319
                     /replace="G/A"
                     /db_xref="dbSNP:rs1407979621"
     variation       2330
                     /replace="T/C"
                     /db_xref="dbSNP:rs1042557255"
     variation       2331
                     /replace="G/A"
                     /db_xref="dbSNP:rs1415897993"
     variation       2332
                     /replace="T/C"
                     /db_xref="dbSNP:rs1468379810"
     variation       2334
                     /replace="A/T"
                     /db_xref="dbSNP:rs906780694"
     variation       2335
                     /replace="C/T"
                     /db_xref="dbSNP:rs1405676262"
     variation       2337
                     /replace="A/G"
                     /db_xref="dbSNP:rs939631450"
     variation       2341
                     /replace="G/T"
                     /db_xref="dbSNP:rs1009700382"
     variation       2343
                     /replace="T/C"
                     /db_xref="dbSNP:rs1598896250"
     variation       2346
                     /replace="G/C"
                     /db_xref="dbSNP:rs1055131242"
     variation       2353
                     /replace="G/A"
                     /db_xref="dbSNP:rs757345703"
     variation       2356
                     /replace="T/C"
                     /db_xref="dbSNP:rs895141295"
     variation       2359
                     /replace="A/G"
                     /db_xref="dbSNP:rs1445789442"
     variation       2362
                     /replace="C/A"
                     /db_xref="dbSNP:rs1347707337"
     variation       2370
                     /replace="A/G"
                     /db_xref="dbSNP:rs1202175849"
     variation       2371
                     /replace="C/G"
                     /db_xref="dbSNP:rs185779796"
     variation       2380
                     /replace="G/C"
                     /db_xref="dbSNP:rs138434008"
     variation       2387
                     /replace="T/C"
                     /db_xref="dbSNP:rs770284764"
     variation       2388
                     /replace="C/A"
                     /db_xref="dbSNP:rs993314617"
     variation       2393
                     /replace="T/C"
                     /db_xref="dbSNP:rs1443952443"
     variation       2401
                     /replace="C/T"
                     /db_xref="dbSNP:rs560721822"
     variation       2402
                     /replace="G/A"
                     /db_xref="dbSNP:rs1359114536"
     variation       2409
                     /replace="C/A/G"
                     /db_xref="dbSNP:rs958909875"
     variation       2410
                     /replace="C/A"
                     /db_xref="dbSNP:rs56338020"
     variation       2418
                     /replace="G/C"
                     /db_xref="dbSNP:rs987794798"
     variation       2423
                     /replace="T/C"
                     /db_xref="dbSNP:rs1397000506"
     variation       2434
                     /replace="-/ATAATTGAATTGAAGTATA"
                     /db_xref="dbSNP:rs1298941130"
     variation       2434
                     /replace="G/A"
                     /db_xref="dbSNP:rs546302489"
     variation       2436..2438
                     /replace="GTG/G"
                     /db_xref="dbSNP:rs1352798528"
     variation       2438
                     /replace="G/A"
                     /db_xref="dbSNP:rs746386287"
     variation       2439
                     /replace="G/T"
                     /db_xref="dbSNP:rs1425783284"
     variation       2440..2442
                     /replace="TTT/TT"
                     /db_xref="dbSNP:rs1387616049"
     variation       2443
                     /replace="C/A"
                     /db_xref="dbSNP:rs571042114"
     variation       2446
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs989165206"
     variation       2450
                     /replace="A/T"
                     /db_xref="dbSNP:rs1457330448"
     variation       2451
                     /replace="C/T"
                     /db_xref="dbSNP:rs1178524164"
     variation       2454
                     /replace="T/C"
                     /db_xref="dbSNP:rs914973532"
     variation       2460
                     /replace="T/C"
                     /db_xref="dbSNP:rs190346392"
     variation       2462
                     /replace="G/A"
                     /db_xref="dbSNP:rs1222597127"
     variation       2464
                     /replace="A/T"
                     /db_xref="dbSNP:rs550407199"
     variation       2465
                     /replace="G/A"
                     /db_xref="dbSNP:rs1469318308"
     variation       2479
                     /replace="C/T"
                     /db_xref="dbSNP:rs1598896277"
     variation       2491
                     /replace="G/T"
                     /db_xref="dbSNP:rs910472367"
     variation       2499
                     /replace="A/T"
                     /db_xref="dbSNP:rs978222171"
     variation       2501
                     /replace="T/C"
                     /db_xref="dbSNP:rs1040243273"
     variation       2502
                     /replace="G/C"
                     /db_xref="dbSNP:rs1473100191"
     variation       2507
                     /replace="A/T"
                     /db_xref="dbSNP:rs892277733"
     variation       2509
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs928161964"
     variation       2511
                     /replace="C/T"
                     /db_xref="dbSNP:rs569123613"
     variation       2513
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs143832275"
     variation       2514
                     /replace="G/A"
                     /db_xref="dbSNP:rs1055792822"
     variation       2519
                     /replace="A/G"
                     /db_xref="dbSNP:rs1598896289"
     variation       2520
                     /replace="G/A"
                     /db_xref="dbSNP:rs759031975"
     variation       2525
                     /replace="C/T"
                     /db_xref="dbSNP:rs1162197821"
     variation       2530
                     /replace="C/T"
                     /db_xref="dbSNP:rs528681249"
     variation       2531
                     /replace="T/C"
                     /db_xref="dbSNP:rs941377486"
     variation       2533
                     /replace="C/T"
                     /db_xref="dbSNP:rs1364696828"
     variation       2539
                     /replace="T/A/C"
                     /db_xref="dbSNP:rs1038405238"
     variation       2545
                     /replace="A/G"
                     /db_xref="dbSNP:rs1249360041"
     variation       2548
                     /replace="T/C"
                     /db_xref="dbSNP:rs1199752843"
     variation       2549
                     /replace="G/A/C"
                     /db_xref="dbSNP:rs1335646473"
     variation       2550
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs1033284093"
     variation       2552
                     /replace="T/G"
                     /db_xref="dbSNP:rs897232976"
     variation       2555
                     /replace="G/A"
                     /db_xref="dbSNP:rs1351319327"
     variation       2567
                     /replace="C/T"
                     /db_xref="dbSNP:rs1262213960"
     variation       2582
                     /replace="G/T"
                     /db_xref="dbSNP:rs994290330"
     variation       2588
                     /replace="C/A"
                     /db_xref="dbSNP:rs182609340"
     variation       2591
                     /replace="C/T"
                     /db_xref="dbSNP:rs147232468"
     variation       2593
                     /replace="C/T"
                     /db_xref="dbSNP:rs1405832715"
     variation       2600
                     /replace="G/C"
                     /db_xref="dbSNP:rs1347418020"
     variation       2601
                     /replace="G/C"
                     /db_xref="dbSNP:rs1008783546"
     variation       2607
                     /replace="T/C"
                     /db_xref="dbSNP:rs1017956044"
     variation       2611
                     /replace="G/T"
                     /db_xref="dbSNP:rs1386636419"
     variation       2614
                     /replace="T/C"
                     /db_xref="dbSNP:rs1354013873"
     variation       2615
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs1024499337"
     variation       2629
                     /replace="T/A"
                     /db_xref="dbSNP:rs1568082929"
     variation       2630..2641
                     /replace="TATATATATATA/TATATATATATATA"
                     /db_xref="dbSNP:rs1220859344"
     variation       2631
                     /replace="A/G"
                     /db_xref="dbSNP:rs1411759519"
     variation       2636
                     /replace="T/C"
                     /db_xref="dbSNP:rs964680280"
     variation       2638
                     /replace="T/C"
                     /db_xref="dbSNP:rs572978123"
     variation       2640
                     /replace="T/A"
                     /db_xref="dbSNP:rs1481233520"
     variation       2641
                     /replace="A/C"
                     /db_xref="dbSNP:rs1021949863"
     variation       2642
                     /replace="A/C"
                     /db_xref="dbSNP:rs1194103788"
     variation       2646
                     /replace="A/G"
                     /db_xref="dbSNP:rs1197566960"
     variation       2651
                     /replace="G/A"
                     /db_xref="dbSNP:rs1447135974"
     variation       2659
                     /replace="T/G"
                     /db_xref="dbSNP:rs1598896318"
     variation       2662
                     /replace="G/A"
                     /db_xref="dbSNP:rs962519213"
     variation       2665
                     /replace="T/C"
                     /db_xref="dbSNP:rs1598896319"
     variation       2668
                     /replace="A/T"
                     /db_xref="dbSNP:rs1203359815"
     variation       2669
                     /replace="T/A"
                     /db_xref="dbSNP:rs972208531"
     variation       2674
                     /replace="A/C"
                     /db_xref="dbSNP:rs1263552246"
     variation       2681
                     /replace="G/A"
                     /db_xref="dbSNP:rs1224311997"
     variation       2693
                     /replace="A/G"
                     /db_xref="dbSNP:rs1325931503"
     variation       2694
                     /replace="C/T"
                     /db_xref="dbSNP:rs764468540"
     variation       2705
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs1232601126"
     variation       2711
                     /replace="T/A"
                     /db_xref="dbSNP:rs1320828506"
     variation       2712
                     /replace="T/C"
                     /db_xref="dbSNP:rs952067507"
     variation       2718
                     /replace="T/C"
                     /db_xref="dbSNP:rs986146508"
     variation       2729
                     /replace="G/T"
                     /db_xref="dbSNP:rs910524939"
     primer_bind     complement(2736..2778)
                     /label=Ts18 5' into 4.721_rev
     variation       2736
                     /replace="C/T"
                     /db_xref="dbSNP:rs944633403"
     misc_feature    complement(2755..2758)
                     /label=961rev
                     /note="MIT-Specificity score: 83, Efficiency Doench2016 = 
                     66, Efficiency Moreno-Mateos = 52, guide sequence: 
                     GCATTCCTCTCTCTAATCAG, full details/primers at 
                     http://crispor.org/crispor.py?batchId=7eWCVYkTVCNV1Z1Md6Da"
     promoter        2772..4496
                     /label=CAGG
     enhancer        2772..3151
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3153..3430
                     /label=chicken beta-actin promoter
     intron          3431..4447
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     CDS             3431..3523
                     /gene="exon 1"
                     /product="Chicken b-actin splice donor from exon 1"
                     /label=exon 1
                     /note="/SECDrawAs=Gene"
     CDS             3524..4085
                     /gene="(null)"
                     /product="Beta actin intron 1 (lacking 33 most 3' nts)"
                     /label=(null)
                     /note="/SECDrawAs=Gene"
     CDS             4087..4397
                     /gene="(null)"
                     /product="Beta actin intron 1 (lacking 33 most 3' nts)"
                     /label=(null)
                     /note="/SECDrawAs=Gene"
     misc_RNA        complement(4276..4295)
                     /label=cas9 I off94
     misc_RNA        4353..4372
                     /label=Nm Cas9 off82
     misc_RNA        4376..4395
                     /label=cas9 II off91
     CDS             4407..4447
                     /gene="(null)"
                     /product="Rabbit beta-globin intron (3' part of)"
                     /label=(null)
                     /note="/SECDrawAs=Gene"
     CDS             4448..4496
                     /gene="(null)"
                     /product="rabbit b-globin 3rd exon (5' part of)"
                     /label=(null)
                     /note="/SECDrawAs=Gene"
     unsure          4490..4509
                     /label=cas12a III off97
     misc_RNA        complement(4491..4513)
                     /label=cas12a II off95
     unsure          4491..4510
                     /label=cas12a IV off95
     unsure          4493..4512
                     /label=cas12a I off95
     primer_bind     4597..4616
                     /label=Ts18 5' into  4.721_fwd
     protein_bind    join(4632..4656,4657..4658,4659..4683)
                     /label=BxbI attP - CT
                     /label=BxbI attP
                     /bound_moiety="BxbI integrase"
                     /note="attP site for the BxbI integrase (Ghosh, P et.al.; 
                     2003)
                     central di-nucleotide mutated to cT
                     "
     CDS             4696..5934
                     /codon_start=1
                     /label=2xFKBP12-Casp9
                     /translation="MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDR
                     NKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV
                     ELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRT
                     RTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHG
                     CQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFE
                     VASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRD
                     PKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKT
                     SVDYPYDVPDYALD"
     CDS             4705..5025
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             5902..5928
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     Site            5935..6000
                     /label=gsgP2A
                     /site_type="cleavage site"
     CDS             5944..6000
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6001..6399
                     /label=Bsd
     protein_bind    join(6409..6433,6434..6435,6436..6460)
                     /label=Bxb1 attP - GT
                     /label=Bsxb1 attP
                     /bound_moiety="BxbI integrase"
                     /note="attP site for the BxbI integrase (Ghosh, P et.al.; 
                     2003)"
     polyA_site      6467..6663
                     /label=SV40
     polyA_signal    6491..6572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6645..6683
                     /label=Ts18 3' into 4.721_fwd
     misc_feature    complement(6664..6680)
                     /label=961rev
                     /note="MIT-Specificity score: 83, Efficiency Doench2016 = 
                     66, Efficiency Moreno-Mateos = 52, guide sequence: 
                     GCATTCCTCTCTCTAATCAG, full details/primers at 
                     http://crispor.org/crispor.py?batchId=7eWCVYkTVCNV1Z1Md6Da"
     variation       6667
                     /replace="A/C"
                     /db_xref="dbSNP:rs966430182"
     variation       6670
                     /replace="G/C"
                     /db_xref="dbSNP:rs1171703235"
     variation       6672
                     /replace="G/A"
                     /db_xref="dbSNP:rs1255119893"
     variation       6681
                     /replace="T/G"
                     /db_xref="dbSNP:rs369692846"
     variation       6683
                     /replace="C/T"
                     /db_xref="dbSNP:rs1478641781"
     variation       6685
                     /replace="G/C"
                     /db_xref="dbSNP:rs1190875623"
     variation       6686
                     /replace="G/A"
                     /db_xref="dbSNP:rs977814011"
     variation       6695
                     /replace="C/T"
                     /db_xref="dbSNP:rs913771318"
     variation       6712
                     /replace="A/C"
                     /db_xref="dbSNP:rs1598896336"
     variation       6715
                     /replace="C/A/T"
                     /db_xref="dbSNP:rs945213733"
     variation       6717
                     /replace="C/T"
                     /db_xref="dbSNP:rs754005958"
     variation       6720
                     /replace="C/T"
                     /db_xref="dbSNP:rs1459856748"
     variation       6728
                     /replace="C/T"
                     /db_xref="dbSNP:rs1043529617"
     variation       6729
                     /replace="G/A"
                     /db_xref="dbSNP:rs903618994"
     variation       6737
                     /replace="A/G"
                     /db_xref="dbSNP:rs1340103356"
     variation       6743
                     /replace="C/T"
                     /db_xref="dbSNP:rs1422405867"
     variation       6744
                     /replace="A/G"
                     /db_xref="dbSNP:rs1272035282"
     variation       6747
                     /replace="A/G"
                     /db_xref="dbSNP:rs928120668"
     variation       6749
                     /replace="C/A"
                     /db_xref="dbSNP:rs961174942"
     variation       6750
                     /replace="C/T"
                     /db_xref="dbSNP:rs553483482"
     variation       6758
                     /replace="C/T"
                     /db_xref="dbSNP:rs991064410"
     variation       6763
                     /replace="G/A"
                     /db_xref="dbSNP:rs1598896346"
     variation       6766
                     /replace="T/C"
                     /db_xref="dbSNP:rs916801707"
     variation       6771
                     /replace="A/G/T"
                     /db_xref="dbSNP:rs896138295"
     variation       6778
                     /replace="T/A"
                     /db_xref="dbSNP:rs1014498665"
     variation       6779
                     /replace="C/T"
                     /db_xref="dbSNP:rs1024969166"
     primer_bind     complement(6781..6798)
                     /label=Ts18_into_eGxx_rev
     variation       6783
                     /replace="C/T"
                     /db_xref="dbSNP:rs1415528459"
     variation       6799
                     /replace="G/C"
                     /db_xref="dbSNP:rs1410206033"
     variation       6804
                     /replace="T/C"
                     /db_xref="dbSNP:rs898042729"
     variation       6814
                     /replace="G/C"
                     /db_xref="dbSNP:rs994075210"
     variation       6820
                     /replace="C/T"
                     /db_xref="dbSNP:rs578179316"
     variation       6821
                     /replace="A/T"
                     /db_xref="dbSNP:rs941623681"
     variation       6824
                     /replace="C/T"
                     /db_xref="dbSNP:rs757507368"
     variation       6833
                     /replace="T/A/C"
                     /db_xref="dbSNP:rs1038373813"
     variation       6836
                     /replace="A/G"
                     /db_xref="dbSNP:rs1428963574"
     variation       6838
                     /replace="A/G"
                     /db_xref="dbSNP:rs1312740365"
     variation       6839
                     /replace="T/C"
                     /db_xref="dbSNP:rs918632758"
     variation       6842
                     /replace="G/T"
                     /db_xref="dbSNP:rs1027797252"
     variation       6845
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs625628"
     variation       6858
                     /replace="T/C"
                     /db_xref="dbSNP:rs796496054"
     variation       6865
                     /replace="G/A"
                     /db_xref="dbSNP:rs1187061355"
     variation       6869
                     /replace="G/A"
                     /db_xref="dbSNP:rs986200297"
     variation       6875
                     /replace="A/-"
                     /db_xref="dbSNP:rs747030292"
     variation       6877
                     /replace="A/G"
                     /db_xref="dbSNP:rs1598896361"
     variation       6879
                     /replace="T/C"
                     /db_xref="dbSNP:rs1203096706"
     variation       6882
                     /replace="G/A"
                     /db_xref="dbSNP:rs1341631177"
     variation       6884
                     /replace="T/A/C"
                     /db_xref="dbSNP:rs563743212"
     variation       6890
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs1051626597"
     variation       6901
                     /replace="T/G"
                     /db_xref="dbSNP:rs1598896367"
     variation       6906
                     /replace="C/G/T"
                     /db_xref="dbSNP:rs750505258"
     variation       6909
                     /replace="T/C"
                     /db_xref="dbSNP:rs758458450"
     variation       6914
                     /replace="T/C"
                     /db_xref="dbSNP:rs1009126249"
     variation       6919
                     /replace="T/C"
                     /db_xref="dbSNP:rs1555662624"
     variation       6922
                     /replace="C/T"
                     /db_xref="dbSNP:rs1038894977"
     primer_bind     complement(6926..6964)
                     /label=Ts18 3' into 4.721_rev
     variation       6926
                     /replace="G/A"
                     /db_xref="dbSNP:rs1598896370"
     variation       6928
                     /replace="A/C"
                     /db_xref="dbSNP:rs138725483"
     variation       6945
                     /replace="C/T"
                     /db_xref="dbSNP:rs1326508386"
     primer_bind     7074..7093
                     /label=Ts18 5' into  4.721_fwd
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ctgggcccag ccggccagat ctgagctcgc ggccgcgatg
     1921 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1981 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttt caatactgca
     2041 tgagttccgc aaataaacca tcccactcac catcactgtt catgcatttc ctacaacagc
     2101 ctatatctag ttcatccatg tccgcttcta tcttttctct cccaggtttc tgcagcaatc
     2161 atactctcat ttctttctag gggctctaac ttctgcttct ctgcttgtag gcctatgtcc
     2221 ttctcttctg ttaatgacat tgggcttttc cccctcttta tgcacctgca gtcaaggtgg
     2281 gaaacaaaaa gattcgaaac tattttgact cagacctagg aacttgtaat gtcaccataa
     2341 gatgagattc ctgtctccag ccaaatctca ccttgaattg taataatccc tatgtgtcaa
     2401 cggaggagcc aggtagagat aattgaatta taggggtggt ttcccccata ctgttctcat
     2461 ggtagtgaat aagtctcaca agatctgatg gttctataaa tggaagatcc cccgcacaag
     2521 ctctcttgcc tgctaccatg taagacatgc ctttgcttct cctttacctt ctgccatgat
     2581 tgtaaggcct ccccagccag gtggaattgt gagtccatta agcctctttt atatatatat
     2641 aaattaccca gtctcagata tgtctttatt aacagtgtga gaacagacta caacctgacc
     2701 ctgacaatcc ttgtgcttcc ctcatcttgc ctcttctatt tccccactct gcctctgaat
     2761 catcgtcgat cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2821 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2881 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2941 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     3001 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     3061 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     3121 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
     3181 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
     3241 ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg
     3301 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
     3361 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
     3421 gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc
     3481 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
     3541 cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg
     3601 gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg
     3661 gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg
     3721 ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag
     3781 cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt
     3841 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc
     3901 ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg
     3961 tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc
     4021 gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg
     4081 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg
     4141 cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg
     4201 ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat
     4261 gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg
     4321 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
     4381 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
     4441 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
     4501 ctcgaggact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4561 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttaatt
     4621 cccggggatc cgtggtttgt ctggtcaacc accgcgctct cagtggtgta cggtacaaac
     4681 ccacacgtgg ccaccatgct cgagggagtg caggtggaga ctatctcccc aggagacggg
     4741 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
     4801 ggaaagaaag ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
     4861 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
     4921 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
     4981 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaatctgg cggtggatcc
     5041 ggagtcgacg gatttggtga tgtcggtgct cttgagagtt tgaggggaaa tgcagatttg
     5101 gcttacatcc tgagcatgga gccctgtggc cactgcctca ttatcaacaa tgtgaacttc
     5161 tgccgtgagt ccgggctccg cacccgcact ggctccaaca tcgactgtga gaagttgcgg
     5221 cgtcgcttct cctcgctgca tttcatggtg gaggtgaagg gcgacctgac tgccaagaaa
     5281 atggtgctgg ctttgctgga gctggcgcgg caggaccacg gtgctctgga ctgctgcgtg
     5341 gtggtcattc tctctcacgg ctgtcaggcc agccacctgc agttcccagg ggctgtctac
     5401 ggcacagatg gatgccctgt gtcggtcgag aagattgtga acatcttcaa tgggaccagc
     5461 tgccccagcc tgggagggaa gcccaagctc tttttcatcc aggcctgtgg tggggagcag
     5521 aaagaccatg ggtttgaggt ggcctccact tcccctgaag acgagtcccc tggcagtaac
     5581 cccgagccag atgccacccc gttccaggaa ggtttgagga ccttcgacca gctggacgcc
     5641 atatctagtt tgcccacacc cagtgacatc tttgtgtcct actctacttt cccaggtttt
     5701 gtttcctgga gggaccccaa gagtggctcc tggtacgttg agaccctgga cgacatcttt
     5761 gagcagtggg ctcactctga agacctgcag tccctcctgc ttagggtcgc taatgctgtt
     5821 tcggtgaaag ggatttataa acagatgcct ggttgcttta atttcctccg gaaaaaactt
     5881 ttctttaaaa catcagtcga ctatccgtac gacgtaccag actacgcact cgacggaagc
     5941 ggagccacta acttctccct gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca
     6001 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     6061 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     6121 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     6181 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     6241 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     6301 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     6361 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaat aaggtaccgt ggtttgtctg
     6421 gtcaaccacc gcggtctcag tggtgtacgg tacaaaccca accggtgttt ggacaaacca
     6481 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     6541 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     6601 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     6661 gtaattagag agaggaatgc ttctggggta actgctaatt gaaagtaggt tagtctcttc
     6721 cacatagcga gagctaatca cccatgatcc acaattccag gagcatgaac atgtatctcc
     6781 ctcaccagcc tgatgcctga aattcaaatg caagagattc attccttgaa attagatatt
     6841 ggctctaaag attcttttaa agtgggaggt ggccagaatg agatgattgc tatagtcagg
     6901 tttaacaatc aactgaaata ccccagaatc tgaaaaggtt acctcatgca agcttggcac
     6961 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     7021 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     7081 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     7141 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     7201 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     7261 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     7321 tcagaggttt tcaccgtcat caccgaaacg cgcga
//
