LOCUS       sequence-471660-        8377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mNeonGreen^NSEC^ repair template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8377)
  TITLE     Nested Self Excising Cassette (NSEC) repair templates
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8377)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pTG610
FEATURES             Location/Qualifiers
     source          1..8377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1260..1282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1382..1401
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1595..1617
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1609..1626
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1610..1626
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      2074..2083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2704..2737)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2851..3876)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    complement(7164..7197)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(7387..7403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7387..7403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7400..7422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7411..7427
                     /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(join(7435..7441,7442..7459,7460..7465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7480..7501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7618..7635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7789..8377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7869..7888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gcccttttgt ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagactgtc acagcttgtc tgtaagcgga tgccgggagc
     1321 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     1381 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     1441 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     1501 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1561 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1621 gccagtgaat tgacgcgtat tgggatatcc caatgcacta gtcgtgctaa cgatatgtct
     1681 gaacaagaga actgcgagcc taggaaggag tctggatctg tttcctccga gcaacttgct
     1741 caattccgtt ccctcgacgg agcatcgatg gtctccaagg gagaggagga caacatggcc
     1801 tccctcccag ccacccacga gctccacatc ttcggatcca tcaacggagt cgacttcgac
     1861 atggtcggac aaggaaccgg aaacccaaac gacggatacg aggagctcaa cctcaagtcc
     1921 accaaggtaa gtttaaacat atatatacta actaaccctg attatttaaa ttttcaggga
     1981 gacctccaat tctccccatg gatcctcgtc ccacacatcg gatacggatt ccaccaatac
     2041 ctcccatacc cagacggaat gtccccattc caagccgcca tggtcgacgg atccggatac
     2101 caagtccacc gtaccatgca attcgaggac ggagcctccc tcaccgtcaa ctaccgttac
     2161 acctacgagg gatcccacat caaggtaagt ttaaacagtt cggtactaac taaccataca
     2221 tatttaaatt ttcagggaga ggcccaagtc aagggaaccg gattcccagc cgacggacca
     2281 gtcatgacca actccctcac cgccgccgac tggtgccgtt ccaagaagac ctacccaaac
     2341 gacaaggtaa gtttaaacat gattttacta actaactaat ctgatttaaa ttttcagacc
     2401 atcatctcca ccttcaagtg gtcctacacc accggaaacg gaaagcgtta ccgttccacc
     2461 gcccgtacca cctacacctt cgccaagcca atggccgcca actacctcaa gaaccaacca
     2521 atgtacgtct tccgtaagac cgagctcaag cactccaaga ccgagctcaa cttcaaggag
     2581 tggcaaaagg ccttcaccga cgtcatggga atggacgagc tctacaagac cggtcaatcc
     2641 ggaaaggtaa gtttaaacaa atatataact taagcgcgca tctgggcctc ccacgaacgg
     2701 gagataactt cgtatagcat acattatacg aagttatgca tgcttagatt tggattgaat
     2761 taatttttta caaagcaaga aaaaaaacac aaagagaata aataaataat aggaaaaaaa
     2821 acaaatattc agtatttaaa atttgaaaat ctactccttg gcccggggcc gcgtcgatgg
     2881 cctcctgttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     2941 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     3001 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     3061 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     3121 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     3181 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     3241 tgttatgcgg ccattgtctg ttagtacgtt gttggagccg aaatccgcgt gcacgaggtg
     3301 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     3361 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     3421 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     3481 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     3541 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     3601 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     3661 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     3721 gctatttacg cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     3781 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     3841 ctcgacagac gtcgcagtca gttcaggctt tttcatatta ccttaaaatt caaaaattaa
     3901 tttcacatca gtcataacca agaaaacaaa ataaacgtta ggacagaaaa ttcaaattaa
     3961 ttaaaaatta tgctgaataa gaaaattaca tgcaaagcct tattttcctc atagaaacag
     4021 ttgaataaca tattttttaa ttgaaaacat agcaaaattc gtggtaaaat ttgtcaaaac
     4081 cttggaaaaa atcgaaaaaa gaacatttcc cgatgcattt tcctatttct cgttgcctga
     4141 caacgtataa acgttgaata ctcactcaac gaatttggct gctggcttgg actctcgcac
     4201 ggaagagacg cgtgtgtaga tttacgacga aagcaaaaat aggccttgaa tctcaaatat
     4261 tttattagaa aacaccaaca atttagatat ttctgtatcc acggttggcg ttgactgctt
     4321 ctgatggtgg agacttgtct gggcactggc agtactcggc atcctttcct ggtcttccgt
     4381 ctggtccagt ctctccctgg gttccgtctg gtccttgacg tccttgcttt ccagatggtc
     4441 cttctggtcc aacggttcct ggctctcctg gtggtccgtg aagaccgttc tgtccagctg
     4501 gtcctgttgg tccttgatct cctcttggtc cctttggtcc tggtcttccg attggatgct
     4561 cggcgtcgcg tcccttctct cctggtggtc ctggctctcc gatctttccg atttgaccgg
     4621 atgatccttg ctctcctgga tgtcctggtt gtccatctct tcctgggttt ccattttgac
     4681 ccctggggcc aggaagtcca cgtggtcctg gtcttccaag agctcctggt ggtccaactg
     4741 gtccttgtgg gcaagtgaag catccgacag actcgcgttg tggagcgata tcatcagctc
     4801 caactcctgg aacaccgtcc aatcctggga ctccatcaag tcctggaact cctggaactc
     4861 cttttggtcc agatggtcca gctggacact tgttatcagc gttgcagtta cacgtaggtc
     4921 catttccatt ggctccattt ggagtctttg gctggttgaa gactggtgga actgctggtg
     4981 gctgtcccgg aggtgcagat ggggatccat cagcatgtgg agttggggta gcgttggtct
     5041 cttcatattg acaacggaca cgcttgctag atcttccgat gacaactatg tccttccaca
     5101 atccattggt agattgtctg aacacattca tctcggtatc aagcttctct ctgaatccat
     5161 caacctccga gtagatggtt gacatggtca tgaagcatac cataagagtg gcgacagtga
     5221 ccgaagcggt gacgtagcaa gccaatttta cagacatctc tgaaattatc aaatttagga
     5281 tttcaaaata attcggccaa agaaatgtaa ctcacctagt cttcgagaag cagagacttg
     5341 tcgatttcct accggattcg tctcctttta ttaggaaatc tttccgcaag ttttcaagtt
     5401 ttttttgata tttatataca ttaccgacga gacacgtgtg cgagacgcag agacgataaa
     5461 agactggata agaacgggag ataaggagta cggatgtagg tgaggaatgt ggccgaaagg
     5521 acagatgggc tttattcggg aagattggtt gggaaatgaa ccagagaaga atgactgaat
     5581 aaagttgttg aaacttggtg agaatggaag atagacaatc gagggcagcg tacgtagaga
     5641 agaagggaat gcttgaaagg attttgcatt tatcttaatc accgtcttcg agaagacgaa
     5701 ccatggcgcc agtttcagaa tccaagtttc taatatagtt catcacaatg ttgacgttgg
     5761 tccaacctcc ggcttgcata atctctggga tggaaactcc agcgcgagcc atgtcacggg
     5821 cagctccgac acgggcggaa tgtccagacc acgctaaata ccgctgtccg gaatcatcct
     5881 tggctccgta gatcaggcga tgagtagctt cgaagattcc ttcaagagct cgagtcgaga
     5941 gttgtgatgt tgcactcggg gcagcaactc cgttcttgcg aacacgacaa aatagataat
     6001 tatttggatc atcggcgact ccggaaaccg aaatccagcg ctcaactagc ttagtcacgc
     6061 ccagggagag cgctttttcg actccggcag tcgagaccag cgtttttgta cgtccgatgt
     6121 ggatgagcat tcttcctccg tctgtgcgac taatatcctt gacgcgaatg cgggcaattt
     6181 cggcgatacg gagaagtgtg ttgtaagcaa ttccgaggaa ggcgaggttt cggatgtcct
     6241 ggcaacgatc ggaattttcc ataagagaac ggacctgatc gaagtcggtt ctgaaaattt
     6301 aaatcagatt agttagttag taaaatcatg tttaaactta cctctcgaat gcaagggctt
     6361 gcttggctct ctctccagca tcaacatttt ccttgcggat acgtctcatt acgagagata
     6421 cggcattaga atctgatggg cgtggaagtc cagatcggcg atggagcata ttgagctgtc
     6481 cgagatgttg ttggatcgtc ttcaccgcca atccacgagc ttggaggtag agcaagtagt
     6541 cacgaacgtc ttctggctcg gctgggaacc acttgcggtt gttaagcttg caccaggcag
     6601 cccaagaacg gcacactgag agaagcatct tccaagtgtg ttccgagaag gcttgtctgt
     6661 cacggaacat atccatgagg ttcttgcgaa cttcatcact tgtcgcgtca actggaagtg
     6721 ctggaagatt ttgatgaaca gtgagaagat ttgacatatt ttcgaagttt tttagatgca
     6781 ctagaacaaa gcgtgttggc ttcctctgag cccgctttcc ttatataccc gcattctgca
     6841 gccttacaga atgttctaga aggtcctaga tgcattcgtt tgaaaatact cccggtgggt
     6901 gcaaagagac gcagacggaa aatgtatctg ggtctcttta ttgtgtacac tacttttcca
     6961 tgtaccgaat gtgagtcgcc ctccttttgc aacaagcagc tcgaatgttc tagaaaaagg
     7021 tggaaaatag tataaatacc gttgaaaata aataccgaac aacatttgct ctaattgtga
     7081 aattagaaat cttcaaacta taatcatgtc actttaccac tatttccgtc cagctcaacg
     7141 ttccgttttt ggtgatctcg cgaataactt cgtatagcat acattatacg aagttataca
     7201 catgaacgag agtctgctat ttaaattttc aggctagcgg aggatctgca tcgggatccg
     7261 gaggcggttc tggcggaggt tctggtgcag gagcctccgg agcctccgga ctagtagggc
     7321 aaatgacatg tccgagcagg aaaattgtga acctaggaaa tcgataacga gcttggcgta
     7381 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7441 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctgtt ccgcttcctc
     7621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     8041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     8101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     8161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     8221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     8281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     8341 caagcagcag attacgcgca gaaaaaaagg atctcaa
//
