LOCUS Exported 4388 bp ds-DNA circular SYN 21-JUL-2022 DEFINITION Knockin of truncated NGFR to target gene. ACCESSION . VERSION . KEYWORDS pTR-339 pUC19-tNGFR-P2A-TIM3 N SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4388) AUTHORS Shy BR, Vykunta V, Ha A, Roth TL, Talbot A, Nguyen DN, Chen YY, Blaeschke F, Vedova S, Mamedov MR, Chung J, Li H, Wolf J, Martin TG, Ye L, Eyquem J, Esensten JH, Alexander M TITLE Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing JOURNAL bioRxiv 2021.09.02.458799 REFERENCE 2 (bases 1 to 4388) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 21, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4388 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(36..58) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 158..177 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 371..393 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 385..402 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 386..402 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1661..1717 /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" primer_bind complement(2174..2190) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2174..2190) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2187..2209) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2198..2214 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(2222..2252) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2267..2288 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2405..2422) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2576..3164) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2656..2675) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3335..4195) /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 3958..3977 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4196..4300) /gene="bla" /label=AmpR promoter primer_bind 4368..4386 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 61 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 121 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 181 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 241 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 301 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 361 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaaatcga gctcggtacc 421 cggggagctc catttcaggt gtatctcatc tgagacatta ttagagggtt ctctttatgt 481 gccatgactg agtagcgttt tcttccactg aagtattgct tcctctttgt tgatatcaca 541 ggacagacat cagaacactt acaggatgtg tgtagtgtgg catgacagag aactttggtt 601 tcctttaatg tgactgtaga cctggcagtg ttactataag aatcactggc aatcagacac 661 ccgggtgtgc tgagctagca ctcagtgggg gcggctactg ctcatgtgat tgtggagtag 721 acagttggaa gaagtaccca gtccatttgg agagttaaaa ctgtgcctaa cagaggtgtc 781 ctctgacttt tcttctgcaa gctccatggg ggcaggtgcc actggccgcg ccatggacgg 841 gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg 901 ccccacaggc ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg 961 tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac 1021 gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct 1081 ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg 1141 ctactaccag gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc 1201 gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg 1261 cacgtattcc gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga 1321 caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc 1381 tggccgttgg attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac 1441 ccaggagcct gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt 1501 gaccacagtg atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat 1561 ccctgtctat tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt 1621 caagaggtgg aacagccgcg ccaagcgctc gggttcgggt gccaccaact tcagcctgct 1681 gaagcaggcc ggcgacgtgg aggagaaccc cggccccttt tcacatcttc cgtttgactg 1741 tgtcctgctg ctgctgctgc tactacttac aagtaagtct cggcatggat atttacaatg 1801 acataatggt gctgtacaga ggacaaggga gagatacaag gatggccatt gtaatgttgt 1861 ttgtaatagc aaaagcttgg aagcaaccta aatgtccgtc gatgagggag tgggtaaata 1921 aatgtgatat aatcatgata caaacagtga gtctgacaaa acatactggt atagaatgtc 1981 tcaaaattat aatgttatta aagaaaaaaa gttgcagaat aatgcatata ttaggcttct 2041 atttgtgtgt gtgcattgtc ggggagcagg aatatgtgga catttcttaa tgtataagct 2101 ctaggaaaag gaaccaggga atggtaaatc ctctagagtc gacctgcagg catgcaagct 2161 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 2221 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 2281 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 2341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 2401 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 2461 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 2521 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 2581 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 2641 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 2701 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 2761 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 2821 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 2881 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 2941 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 3001 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 3061 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 3121 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 3181 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 3241 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 3301 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 3361 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 3421 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 3481 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 3541 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 3601 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 3661 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 3721 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 3781 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 3841 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 3901 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 3961 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 4021 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 4081 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 4141 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 4201 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 4261 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 4321 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 4381 cgaggccc //