LOCUS       sequence-455552-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 1795 retron with split retron ncRNA and gRNA to edit 
            (10bp barcode) EMX1 gene in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  AUTHORS   Khan AG, Rojas-Montero M, Gonzalez-Delgado A, Lopez SC, Fang RF, 
            Crawford KD, Shipman SL
  TITLE     An experimental census of retrons for DNA production and genome 
            editing.
  JOURNAL   Nat Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z.
  PUBMED    39289529
REFERENCE   2  (bases 1 to 6994)
  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"; journalName: "Nat 
            Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z."
COMMENT     Sequence Label: pAGK65
FEATURES             Location/Qualifiers
     source          1..6994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    828..962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(852..871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     906..925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(968..1163)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      982..1117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    1222..1243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1258..1263,1264..1281,1282..1288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1312
                     /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)."
     primer_bind     1301..1323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1320..1336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1320..1336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(1476..1690)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1536..1555)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        complement(2079..2327)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2137..2156)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2307..2327)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    2362..2586
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(2614..2634)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4127..4146)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4154..5170
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4208..4230)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5171..5447)
                     /label=chicken beta-actin promoter
     enhancer        5449..5828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5859..5963
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5964..6032,6033..6824)
                     /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(6182..6201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcggat ccagacatga
      841 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      961 ttagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     1021 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     1081 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     1141 tggttgctga ctaattgaga tgcggatccg ctggcacgac aggtttcccg actggaaagc
     1201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     1261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     1321 aggaaacagc tatgaccatg attacgccaa gctaaaaaaa aaaaagcacc gactcggtgc
     1381 cactttttca agttgataac ggactagcct tatttcaact tgctatgctg tttccagcat
     1441 agctctgaaa cttcttcttc tgctcggact ctttcgtggt ctcatacaga acttataaga
     1501 ttcccaaatc caaagacatt tcacgtttat ggtgatttcc cagaacacat agcgacatgc
     1561 aaatattgca gggcgccact cccctgtccc tcacagccat cttcctgcca gggcgcacgc
     1621 gcgctgggtg ttcccgccta gtgacactgg gcccgcgatt ccttggagcg ggttgatgac
     1681 gtcagcgttc aaaaaaaagc gcgagcggtc gagggaggcc cgaaggcacc accttctcac
     1741 ctggggcacg gtagcctcac cggctctccc ctccggtgag tacctctccg gccggggatg
     1801 acatcgatgt cctccccatt ggcctgcttc gtggcaatgc gccaccggtt gatgtgatgg
     1861 gagaactttc ttgcacgctt tcttctgctc ggactcaggc ccttcctcct ccagcttctg
     1921 ccgtttgttc cccggccgga gaggtactca ccggagggaa gagagccaag aacaggctac
     1981 cttgcggaga gtgtcctgca attcaaccag agcggcgcaa cgccgctctc gcgctcgagg
     2041 cgctgaggct gatggcttgg gacctctctc ggtcgctcgc actttcgtcc tttccacaag
     2101 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
     2161 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
     2221 ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc tctaacagcc
     2281 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcact tagattcagc
     2341 tggatccgat ttcgcagatc tccatagagc ccaccgcatc cccagcatgc ctgctattgt
     2401 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
     2461 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
     2521 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
     2581 gcacaggttt aaactcatta ctaaccggga tcagaccttt cttttcttct ttggtggagc
     2641 ggtctgtctc ttctctaacg cttcgagccg acgcagaaag gctcttcctt tctctgcatc
     2701 tgtcatatga agaaacgcgg cgagtccttt cagttgctcc agcgtctctc cggtaggacc
     2761 tggttttccc tgctcgcggt tatggatggc tgcgcgcaat ctacgcacga cgtcacgggg
     2821 cacccttgcg ccaggaacgc cttctggtgc ttcgttaacc accagtccgg ttaccctttg
     2881 cctagagcct tttctttgca cccgggtttt gtctggatgg agtgtgaagc cttcagcttc
     2941 cagtacacct ttgactctag ccagaagcag cgctacgggg gcgtcggcca ggggaagttc
     3001 cttttgcctt gatttcttgg ctctccgcca gctaaaagta agatcgtcgg catatcgtgt
     3061 gtaagtgaaa cccagcctct tactcagagc tgagagacgc ttatcaagtc tcaggcacag
     3121 agcgttagtg agggcaggag aagtaggagc gccctgtggc agggcgcgag gtcccttagc
     3181 aacgtagagt gtctcacctc taaagcggac cacctcgcgg ggtgcttcag tgctcagtaa
     3241 ggccagcagt gtggccaggt tctctggtaa gccacctttc ctcagcagcc ccttgaccct
     3301 aggccaggtc acactcggaa agaaatcttt catgtcgacc ttgaccacca cgtcagcccc
     3361 ttgatgtgcg agagcgttag tgagtatgct gcgtccagcc acgaacccat gggcagcgcc
     3421 gtgcacgggg agcctctcca ccacattcgc gagaacccag cgttggactg ccttcaattc
     3481 ccgtttgggg gcagtgagtg ttctctttcc gccatccctc ttcggaattt cccaagtctg
     3541 atagtgggtt cccgtgtcaa cttctctgtg gaaactgaac cacctcagcc tggaaacaga
     3601 gatccccagt gctttggcca atgcttcgac ggaatccagt ccttctggca ggccattggc
     3661 tttagcccgc tcctctcgtc ctgcaacgtc gaacttatca gggccgcctg cctcgtccca
     3721 gtgcacgccc acgcccagat gatgaatgtg ggtagccttc catgcttccc aggccagccg
     3781 tttctgggcc cgtctttcgg tggcttcggc cttcttcttt tctttccatg cggccttctg
     3841 tttgtcgctc aggctctcaa aatccacttc ctctgcggcc acgcccttgg ccaccagctg
     3901 ttgtctaacc caagcgtcgg tcccgcccgc ctcttcaata gccttccatc ggagtcgcag
     3961 ggcttcatga tgtgctctcc gggcttcctg tttagcagcg tcgggtcttg taggggcggg
     4021 agcttcggcg cttactgggg gcgcagctgg cacatgggat tcaagtttgg cggtcatggt
     4081 ggcgtaccga tatcgagctc aattctttgc caaaatgatg agacagcaca ataaccagca
     4141 cgttgcccag gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag
     4201 agccgccggt cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc
     4261 cgtccccccg cggacagccc cgaggctgga gagggagaag gggacggcgg cgcggcgacg
     4321 cacgaaggcc ctccccgccc atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc
     4381 cgctagaggg ggtgcggcgg cgcctcccag atttcggctc cgcacagatt tgggacaaag
     4441 gaagtccctg cgccctctcg cacgattacc ataaaaggca atggctgcgg ctcgccgcgc
     4501 ctcgacagcc gccggcgctc cgggggccgc cgcgcccctc ccccgagccc tccccggccc
     4561 gaggcggccc cgccccgccc ggcaccccca cctgccgcca ccccccgccc ggcacggcga
     4621 gccccgcgcc acgccccgta cggagccccg cacccgaagc cgggccgtgc tcagcaactc
     4681 ggggaggggg gtgcaggggg ggttgcagcc cgaccgacgc gcccacaccc cctgctcacc
     4741 cccccacgca cacaccccgc acgcagcctt tgttcccctc gcagcccccc ccgcaccgcg
     4801 gggcaccgcc cccggccgcg ctcccctcgc gcacactgcg gagcgcacaa agccccgcgc
     4861 cgcgcccgca gcgctcacag ccgccgggca gcgcggagcc gcacgcggcg ctccccacgc
     4921 acacacacac gcacgcaccc cccgagccgc tccccccgca caaagggccc tcccggagcc
     4981 cctcaaggct ttcacgcagc cacagaaaag aaacaagccg tcattaaacc aagcgctaat
     5041 tacagcccgg aggagaaggg ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc
     5101 agagccgggg cgggcggcgc gaggcggcgg cggagcgggg cacggggcga aggcagcgcg
     5161 cagcgactcc cgcccgccgc gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg
     5221 ccataaaagg aaactttcgg agcgcgccgc tctgattggc tgccgccgca cctctccgcc
     5281 tcgccccgcc ccgcccctcg ccccgccccg ccccgcctgg cgcgcgcccc cccccccccc
     5341 cgcccccatc gctgcacaaa ataattaaaa aataaataaa tacaaaattg ggggtgggga
     5401 ggggggggag atggggagag tgaagcagaa cgtggggctc acctcgacca tggtaatagc
     5461 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     5521 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     5581 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     5641 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     5701 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     5761 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa
     5821 tcaatgtcga cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5881 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5941 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6001 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6061 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6121 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6181 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6241 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6301 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6361 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6421 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6481 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6541 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6601 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6661 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6721 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6781 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6961 cgtcagaccc cgtagaaaag atcaaaggat cttc
//
