LOCUS       sequence-455374-        6661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-21(+) deriviative with retron Kva1 containing a donor to edit E.
            coli rpoB gene cloned downstream of a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6661)
  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 6661)
  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: pAGD289
FEATURES             Location/Qualifiers
     source          1..6661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     118..137
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        118..136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    137..161
                     /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)."
     CDS             1510..1527
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1580..1598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1594..1641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1678..2133
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1765..1784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1975..1996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2159..2263
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2264..2332,2333..3124)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2482..2501)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3295..3883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3784..3803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4037..4054
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4069..4208
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4134..4153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4294..4316
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4310..4501)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5310..6392)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6354..6373
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6393..6470)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
       61 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
      121 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagga tccgaattcg
      181 aaggagatat acatatgtcc tatatcgatg agctgagcaa gactctgaac tgcactccga
      241 agactattat ctctggctac atcaaggcgc ctaagaaata caaggtgttc tttatcaaaa
      301 agaaaaacgg taaaggcctc cgtgagatcg cgcagccgtc tccggacgtt aagaaagttc
      361 aacgtgctat tgttaacagc ctcctgtctc tcctgccggt tcacgagtgc gcgaccgcgt
      421 atatcgagga aaaatccatt gttgataacg cagaaccgca caagaaaaac gaatacatcc
      481 tcaaaatgga tttcgaaaat tttttctaca gcatcaaacc gcaggatctg aactatttta
      541 tcagcaaaaa cgatattaaa ctgaaccaat ttgaatatga agtaattagc cactacctgt
      601 ttcgtcgcaa gaaaggtgaa aagaaactgc gcctgtgcat cggtgcgccg agctctccga
      661 tgatctccaa catcgttatg tacgacttcg acgaaaaact gttcaactac tgtaaagaaa
      721 acgatgtgat ctacactcgt tacgctgatg acctgtcttt ttcttccgat tcttttgaca
      781 aactgaacga tgtcaaaaac ttcgttatta acctggtgtc tctgcagaag aaaccagaac
      841 tgattatcaa cacttctaaa accaaattta ttggtaaagg tcgtagccgt cgcattactg
      901 gtgtggttat ttctaacacc ggcgatctgg gtgtgggccg ttttctgcgt aagaaaatcc
      961 gcgcactgtt ttacctgtac agcaataaca aaatcaatca taaggacatt ccgtacctcc
     1021 tgggcatgct gtctcacatc aaaaatatcg aaccggacta ttactatagc ctgattaaaa
     1081 ttaacggtga ggacaaactg aaacaactgg gtaaggatag cttcgcagtc ctgcagaaaa
     1141 acaatgacta ataccgttca tatgaggcta gagcaggtga tccataatct tgagacaagc
     1201 agcttgattg atgcaaatca ttaagttagt gtataacata ctctttacgc gatagaggcg
     1261 tatctgcttc atctggtgta tcaccggaac ttgttgattg tggtcattta tgccacggcc
     1321 tccgatttcc gcagcagtga aagagttctt cggttccagc cagctgcctc agtttatgga
     1381 ccagaacaac ccgctgtctg agattacgca caggccgtgc ccttctaggt aaagagcaga
     1441 accccggttt accggggttt tttataacat aagcggctgc cacctgcatg atccgcggcc
     1501 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1561 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1621 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1681 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1741 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1801 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1861 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1921 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1981 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2041 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2101 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2161 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2221 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2281 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2341 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2401 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2461 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2521 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2581 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2641 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2701 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2761 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2821 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2881 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2941 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3001 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3061 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3121 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3181 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3241 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3301 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3361 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3421 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3481 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3541 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3601 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3661 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3721 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3781 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3841 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3901 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3961 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4021 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4081 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     4141 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4201 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4261 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4321 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4381 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4441 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4501 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4561 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4621 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4681 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4741 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4801 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4861 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4921 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4981 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5041 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5101 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5161 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5221 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5281 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5341 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5401 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5461 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5521 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5581 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5641 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5701 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5761 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5821 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5881 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5941 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     6001 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6061 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6121 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6181 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6241 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6301 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6361 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6421 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6481 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6541 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6601 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6661 c
//
